The nitric oxide molecule (NO ·) is without doubt one of the most influential on the pathophysiology of the cardiovascular system. Its effects are broad ranging in this system influencing almost all of its components, particularly the vasculature, platelets and the myocardium. The beneficial effects of this molecule not only in inhibiting the development of cardiovascular disease (CVD) but in minimizing the risk of its acute thrombotic complications makes it an obvious target for therapies designed to enhance its effects. Assessing the tissue effects of NO · in platelets and the myocardium has been the main focus of this thesis. Determinants of platelet responsiveness to nitric oxide in diabetic patients with acute coronary syndromes: Effects of glycaemic control Background: We chose to perform our initial experiments in diabetic patients admitted with an acute coronary syndrome (ACS). This cohort offered us a unique opportunity to assess NO · bioavailability in a group with one of the highest cardiovascular morbidity and mortality rates. We assessed this via platelet NO · responsiveness, which has been shown from our laboratory to be impaired in ACS but to date had not been assessed specifically in a diabetic group. As hyperglycaemia is a known independent predictor of mortality in this group we chose in our first set of experiments to primarily assess the effects of glycaemic control (acute and chronic) on platelet NO · responsiveness. Study: In diabetic patients admitted with an ACS the relationship between glycaemic control and the main determinants of platelet NO · responsiveness, superoxide (O2-)generation and guanylate cyclase activity were assessed. Secondary hypotheses assessed the effects of acute glycaemic control on other potential modulators of NO · responsiveness such as asymmetric dimethylarginine (ADMA), L-arginine, Larginine/ADMA ratio, platelet activated O2- release and C-reactive protein (CRP). Other values assessed included CK rise, non-esterified fatty acid (NEFA) levels, ADP induced platelet aggregation and neutrophil count. We also assessed the determinants of both platelet SNP response and of O2- levels by a stepwise multivariate analysis; parameters assessed were age, sex, blood sugar level (BSL), statin therapy, insulin therapy, ACE-inhibitor therapy and CK elevation. Conclusions: In diabetic patients admitted with an ACS; 1. Admission BSL was inversely correlated with platelet SNP responsiveness and directly correlated with O2- generation, but not with guanylate cyclase activity. 2. Admission BSL also correlated with CK rise, CRP, neutrophil count and ADP enhanced O2- generation. Admission BSL was inversely correlated with ADMA and L-arginine levels. 3. On multivariate analysis, admission BSL was a significant determinant of O2- levels and an inverse determinant of SNP response; with increasing age also a significant inverse determinant of SNP response. Acute Resolution of hyperglycaemia normalizes platelet responsiveness to nitric oxide in diabetics with acute coronary syndromes Background: We then explored whether improving glycaemic control had any effect on platelet NO · responsiveness. This was performed by randomizing diabetics with ACS to either intravenous infusions or subcutaneous injections of insulin. This experiment was designed to determine possible mechanisms to explain the results of the DIGAMI study, which showed that tight glycaemic control decreased mortality in diabetics admitted with an acute myocardial infarction (AMI). Study: Sixty diabetic patients admitted with an ACS were randomized to aggressive IV insulin therapy or standard subcutaneous insulin therapy over 12 hours. The primary objective of this study, was to assess the acute effects of tight glycaemic control on platelet NO · responsiveness, O2- generation and guanylate cyclase activity. To explore all possible mechanisms of any observed effect δ ADMA,L- arginine and Larginine/ADMA ratio, CRP, non esterified fatty acids (NEFA) and platelet aggregation were assessed. Conclusions: In diabetic patients admitted with an ACS; 1. Aggressive glycaemic control resulted in significantly enhanced platelet responsiveness to SNP, related to a reduction in O2- generation. No effect on guanylate cyclase activity was seen. 2. A significant reduction in ADMA and L-arginine levels were observed with intravenous compared to subcutaneous insulin therapy. Effects of an oral glucose load on platelet responsiveness to nitric oxide Background: Elevated BSLs are associated with adverse cardiovascular outcomes in ACS in both diabetic and non-diabetic subjects. Hyperglycaemia is no longer considered an ‘ innocent bystander ’ however, having detrimental mechanistic implications on a number of protective biological systems. Indeed, harmful effects of sudden glucose loads such as post-prandial hyperglycaemia on the cardiovascular system are becoming apparent. While our previous experiments had focused on the impact of hyperglycaemia and its subsequent correction, on NO · bioavailability, our follow up experiments assessed the effect of an oral glucose load in normal subjects and patients with known cardiovascular disease. In light of the previous findings, we assessed the effects of an acute glucose load on platelet NO · responsiveness and vascular reactivity as assessed by applanation tonometry. Study: 8 healthy and 10 ‘ high-risk ’ cardiovascular subjects were enrolled into this study. A 75 gm glucose load was administered and baseline and 2 hour data were assessed. Platelet SNP responsiveness and O2- generation along with augmentation index (AIx) and ADP-induced platelet aggregation were assessed in all subjects. In the ‘ high-risk ’ cohort cGMP generation and insulin levels were also evaluated. Conclusions: In the healthy volunteers; 1. No significant change occurred in any of the variables assessed In the ‘ high-risk ’ cohort 1. 80 % of patients had undiagnosed impaired glucose handling 2. A significant increase in soluble guanylate cyclase activity was associated with an oral glucose load possibly related to an increase in insulin levels Lack of inotropic effect of nitric oxide in rat papillary muscle. Background: Our final set of experiments moved away from platelet studies to the assessment of myocardial contractility. Differing methodologies and animal models have resulted in variable results in the assessment of the inotropic effects of NO · . While the consensus is that NO · donors have a positive inotropic effect in low doses and are negatively inotropic at high doses, this has been difficult to show in the papillary muscle of most animal models. We addressed this issue in the papillary muscle of the Sprague-Dawley rat. Study: The experimental protocol involved the assessment of NO · effects on contractility on the left ventricular muscle of the Sprague-Dawley rat. These studies initially involved assessing the most stable preparation, comparing a 10bpm with a 35bpm protocol. The effect of the NO · donor SNP was then assessed not only on the isolated LV papillary muscle but also in the presence of β -adrenoceptor stimulation and an ischaemic/reperfusion model (15mins of anoxia/ 30 mins of reperfusion). Conclusions: In the left ventricular papillary muscle of the Sprague-Dawley rat 1. A stimulation frequency of 10bpm resulted in a more stable preparation over a 60 minute protocol, compared to 35bpm. 2. The isolated rat papillary muscle had no measurable response to exogenous NO · donors. The inotropic effects of β -adrenoceptor stimulation and ischaemia-reperfusion were NO · -independent in this model. While the field of NO · research is rapidly expanding, we forget that it is only just over 20 years since we were first aware of its existence. From Furchgott and Zawadski ' s ( Furchgott and Zawadzki, 1980 ) initial observation of an endotheliumderived relaxing factor ( EDRF ) to the subsequent studies by Palmer et al. ( Palmer et al., 1987 ) and Ignarro et al. ( Ignarro et al., 1987 ) showing that EDRF was indeed NO ·, many questions remain unanswered in relation to the 1992 ‘ molecule of the year ’. This thesis contributes to our understanding of NO · and its associated bioavalability in a number of tissues, particularly in relation to a high-risk cohort, the hyperglycaemic diabetic with an ACS. / Thesis (Ph.D.)--Medical School, 2004.
| Identifer | oai:union.ndltd.org:ADTP/263868 |
| Date | January 2004 |
| Creators | Worthley, Matthew I |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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