No description available.

Spinal cord stimulation (SCS) represents an acceptable treatment modality for patients with chronic angina pectoris refractory to standard therapy, but its mechanism of action remains unclear. To develop an experimental paradigm to study this issue, ameroid (AM) constrictors were implanted around the left circumflex coronary artery (LCx) in canines. Six weeks later, unipolar electrograms were recorded from 191 sites in the LCx territory in the open-chest, anesthetized state under basal pacing at 150 beats/min. We investigated the effect of SCS on ST segment displacements induced in the collateral-dependent myocardium in response to two stressors: (i) transient bouts of rapid ventricular pacing (TRP: 240/min for 1 min) and (ii) angiotensin II administered to right atrial neurons via their coronary artery blood supply. ST segment responses to TRP consisted of ST segment elevation in central areas of the LCx territory and ST depression at more peripheral areas. Such responses were unchanged when TRP was applied under SCS. Shortening of repolarization intervals in the metabolically compromised myocardium in response to TRP was also unaffected by SCS. In contrast, ST segment responses to intracoronary angiotensin II, which consisted of increased ST elevation, were attenuated by SCS in 6/8 preparations. The modulator effects of SCS were greatest at sites at which the greatest responses to angiotensin II occurred in the absence of SCS. These data indicate that spinal cord stimulation may attenuate the deleterious effects that stressors exert on the myocardium with reduced coronary reserve, particularly stressors associated with chemical activation of the intrinsic cardiac nervous system.

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT1 receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPβS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.

No description available.

No description available.

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Introduction: Obesity, which is implicated in the development of the metabolic syndrome (MS) is reaching epidemic proportions worldwide. MS significantly increases the risk of developing cardiovascular disease, which includes coronary artery disease. The current absence of animal models of diet induced obesity and the MS makes the investigation of the cardiovascular consequences of MS virtually impossible. As a result the effects of the MS on cardiac function, morphology and susceptibility to ischaemia are not well understood. Aims: We set out to: 1) develop and characterize a rodent model of dietinduced obesity and the MS, 2) investigate the susceptibility of hearts from these animals to ischaemia/reperfusion induced injury and, 3) determine whether angiotensin II (Ang II) and endothelin-1 (ET-1) plays a role in cardiac remodelling and/or the severity of ischaemia and reperfusion injury in this model. Methods: Male Wistar rats were fed a standard rat chow diet or cafeteria diet (CD) for 16 weeks. After the feeding period rats were sacrificed and blood and myocardial tissue samples were collected to document biochemical changes in these animals. Hearts were perfused on the isolated working rat heart perfusion apparatus to assess myocardial mechanical function before and after ischaemia. In a separate series of experiments, hearts underwent coronary artery ligation to determine the incidence and duration of ventricular arrhythmias during ischaemia and reperfusion, using electrocardiography. To assess a possible link between myocardial remodelling and ischaemia/reperfusion injury and myocardial Ang II and ET-1 content, we also measured these peptides under basal conditions and during ischaemia. Two-dimensional targeted Mmode echocardiography was used to assess in vivo myocardial mechanical function in control and obese rats. Results: After 16 weeks on the CD, obese rats satisfied the World Health Organization (WHO) criteria for the MS by having visceral obesity, insulin resistance, dyslipidaemia and an elevated systolic blood pressure, compared to control rats. Circulating Ang II levels, but not ET-1 levels, were elevated in CD fed rats. Obese rats had cardiac hypertrophy and ex vivo basal myocardial mechanical function was depressed in the CD fed rat hearts compared to control rat hearts. CD fed rat hearts had poorer aortic output (AO) recoveries compared to hearts from control rats. These hearts also had a higher incidence and duration of reperfusion arrhythmias. No such functional differences were seen in the in vivo experiments. No differences in basal or ischaemic myocardial Ang II and ET-1 levels were seen in either group. Conclusion: We have developed and characterized a model of diet-induced obesity and the MS. Obesity is associated with cardiac hypertrophy and an increased myocardial susceptibility to ischaemia and reperfusion injury in our model. The hearts from obese rats were also more prone to reperfusion ventricular arrhythmias. As myocardial function was only poorer in the ex vivo obese animal experiments, our data suggests that the obesity associated changes in function observed in the ex vivo studies may be related to the absence of circulating substrates or factors, which are essential for their normal mechanical function.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / Insulin stimulates the production of nitric oxide (NO) in endothelial cells and cardiac myocytes by a signalling pathway that involves the insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Physiological concentrations of NO play an important part in maintaining normal vascular function. It has been suggested that nitric oxide synthase (NOS) activity and NO production are chronically impaired in diabetes mellitus by an unknown mechanism. The reninangiotensin system and subsequent production of angiotensin II (Ang II) are elevated in obesity and diabetes while antagonism of the AT1 receptor with Losartan has beneficial effects in patients with insulin resistance and type II diabetes. Aims: We therefore aimed to investigate (i) the effect of Ang II on myocardial insulin signalling with regards to key proteins (IRS-1, PKB/Akt, eNOS and p38 MAPK) in correlation with NO production, (ii) the effect of Losartan on these parameters. Methods: Hyperphagia-induced obese, insulin resistant rats (DIO=diet supplemented with sucrose and condensed milk) were compared to age-matched controls. Half the animals were treated with 10mg/kg Losartan per day for 1 week. Isolated hearts were perfused with or without 0.03 μIU/mL insulin for 15 min. Blood glucose, bodyweight, intraperitoneal fat and plasma insulin and Ang II were recorded. Proteins of interest and their phosphorylation were determined by Western blotting. NO production was flow cytometrically analyzed. ANOVA followed by the Bonferroni correction was used with a p< 0.05 considered significant. Results: DIO animals had significant elevated bodyweight, blood glucose, plasma insulin and Ang II levels. Our data showed that the hearts from the DIO animals are insulin resistant, ultimately reflected by the attenuated activation of the key proteins (IRS-1, PKB/Akt and eNOS) involved in insulin signalling as well as NO production. AT1 receptor antagonism improved NO production in isolated adult ventricular myocytes from DIO animals while concurrently enhancing expression of eNOS, PKB/Akt and p38 MAPK. In contrast, NO production as well as expression of eNOS and PKB/Akt was attenuated in control animals after Losartan treatment. Conclusion: These results suggested that Ang II via AT1 or AT2 receptors, modulates protein expression of both PKB/Akt and eNOS. This encouraged us to investigate the involvement of AT2 receptors in the observed changes. To investigate this we needed to establish a culture of neonatal rat cardiac myocytes treated with raised fatty acids and Ang II. If similar changes were induced as observed in the hearts of DIO animals, the involvement of the AT1 and AT2 receptors could be investigated using specific antagonists against these receptors. Primary cultured ventricular myocytes were isolated from 1-3 day old Wistar rat pups. They were cultured for 48 hours before the addition of palmitate and oleate at a concentration of 0.25 mM each and were treated with or without the fatty acids for a period of 4 days. After 18 hours of serum starvation, cells were stimulated with or without 10 nM insulin for 15 minutes. The effect of fatty acid treatment on cell viability and glucose uptake were assessed by trypan blue and propidium iodide staining and 2-deoxy-D-3[H] glucose uptake respectively. Protein levels and phosphorylation of key proteins (PKB/Akt, PTEN and p38 MAPK) in insulin signalling was determined by Western blotting. 0.25 mM Fatty acids did not result in the loss of cell viability. Contrary to expectation, fatty acid treatment led to enhanced basal glucose uptake but lower Glut 1 protein expression. Basal protein expression of PPARα was, however, upregulated as was the expression of the phosphatase, PTEN. The latter could explain the lower PKB/Akt phosphorylation also documented. From these results we conclude that neonatal cardiac myocytes, cultured in the presence of elevated fatty acids, did not respond in a similar manner as the intact hearts of our animals and further modifications of the system might be needed before it can be utilized as initially planned.

<p>The kidney regulates the excretion of water and electrolytes, which maintains homeostasis and enables control of arterial blood pressure. Hyaluronan, a large negatively charged interstitial glucosaminoglycan, is heterogeneously distributed within the kidney, primarily found in the medulla.</p><p>Medullary hyaluronan content changes depending on the state of body hydration and plays a part in fluid regulation through its water binding and viscoelastic properties. </p><p>The aim of this thesis was to provide new insight into the regulation of intrarenal hyaluronan during different states of body hydration, during completion of kidney development, and during diabetes mellitus.</p><p>Dehydration reduces medullary interstitial hyaluronan in parallel with reduced hyaluronan synthase 2 gene expression and increased urinary hyaluronidase activity. Acute hydration results in an increase in medullary hyaluronan, an increase that requires nitric oxide and prostaglandins. Urinary hyaluronidase activity decreases during hydration. The elevation of hyaluronan is important for reducing water permeability of the interstitium i.e. favoring diuresis.</p><p>Changes in hyaluronan concentration constitute a morphoregulatory pathway that plays a key role in nephrogenesis. The reduction in neonatal hyaluronan depended on an angiotensin II mediated process that does not appear dependent on lymph vessel formation. If angiotensin II is blocked with an ACE inhibitor, hyaluronan accumulates, which results in structural and functional abnormalities in the kidney. </p><p>Renomedullary hyaluronan is elevated during uncontrolled diabetes, which coincides with induction of hyaluronan synthase 2 mRNA, hyperglycemia, glucosuria, proteinuria and overt diuresis. The levels of hyaluronan are probably at a <i>terminus ad quem</i> as no further response was seen during hydration. The higher interstitial expression of hyaluronan during diabetes may be involved in the progression of diabetic nephropathy.</p><p>This thesis in physiology provides new mechanistic insights into the regulation of renal hyaluronan during various aspects of fluid handling.</p>

Hypertension, or high blood pressure (BP), is an ever-growing epidemic in the developing world. Understanding the genetics behind essential hypertension (EH), or hypertension with no known cause, is especially important. In this study, three single nucleotide polymorphisms (SNPs) known to be linked to an increase in susceptibility to EH were quantified from a cohort of Kenyans living in the Kasigau region. The SNPs are located in three genes that are part of the renin angiotensin system, the primary regulatory pathway in humans controlling BP. They include: AGT (rs699), AGTR1 (rs5186), and HSD11β2 (rs5479). Overall, by using a fluorescent-based RT-PCR technique, the genotype distribution of AGT (rs699) was 0.63 C/C, 0.34 C/T, and 0.03 T/T. When evaluated as normotensive, prehypertensive, Stage I, or Stage II categories the allele frequencies for f(C)= 0.77,0.85,0.81, 0.77, respectively, and demonstrated Hardy Weinberg Equilibrium (HWE) as assessed by Χ2, p < 0.05. The genotype distribution of AGTR1 (rs5186) was 0.96 A/A, 0.03 A/C, and 0.00 C/C and the genotype distribution of HSD11β2 (rs5479) was 0.46 A/A, 0.46 A/C, and 0.08 C/C. The majority of genotype frequencies for each SNP were in HWE, with the exception of the AGT (rs699) SNP found in the sublocation of Bughuta suggesting other evolutionary selective pressures may be at work in this subpopulation. The high prevalence of the susceptible C allele for AGT (rs699) likely implies it is a critical factor in the high prevalence of EH observed in this population.