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Title: Molecular pharmacology of altered cardiopulmonary function in inflammation
Author: El-Awady, Mohammed
ISNI:       0000 0004 2675 1400
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2008
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Inflammation has incompletely characterized effects on cardiopulmonary vascular reactivity. Sepsis is a major inflammatory disease characterized by two main vasomotor complications, generalized vasodilation with hyporesponsiveness to vasoconstrictors and pulmonary hypertension. The main aim of this study is to examine the molecular mechanisms involved in cardiopulmonary vascular reactivity changes induced by the powerful inflammatory stimulus lipopolysaccharide (LPS). Pulmonary and aortic rings from male Wistar rats (250-300g) were isolated and incubated for 20 h in culture medium (DMEM+10% FBS) with or without LPS (E. coli O55:B5, 10 μ⁻¹). The effect of organ culture and LPS type, concentration and incubation time in addition to tissue contraction to endothelin-1 (ET-1), phenylephrine, 80 mM KCl, and U46619; and relaxation responses to ACh, sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY 41-2272, T-0156, nifedipine, SKF-96365, Ro-31-8425, and Y-27632 were measured by standard organ bath techniques. Nitric oxide (NO) production was measured by the Griess method and SNP-induced cGMP production was measured by ELISA. mRNAs expression levels of eNOS, iNOS, ET-1, ETA and ETB were measured by qRT-PCR and the expression levels of PKC, sGCα₁, sGCβ₁ and PDE5 and phosphorylation of MLC₂₀, ROKα, CPI-17 and MYPT1 were measured by immunoblotting. The effect of endothelium removal, indomethacin, trolox, external Ca²⁺ removal, 1400W, ODQ, glibenclamide, iberiotoxin and cycloheximide in addition to changes in intracellular Ca²⁺ ([Ca²⁺]i) in aortic vascular smooth muscle cells (VSMCs) induced by ET-1 were also measured. LPS selectively induced vascular hyporeactivity to different vasoconstrictors in rat aorta but not in the pulmonary artery, which is not due to organ culturing and is not affected by changing the LPS type, but is enhanced by increasing LPS concentration or the incubation time. This aortic hypocontractility to ET-1 is largely mediated by NO-independent activation of sGC and depends on external Ca²⁺ influx through non-VOCCs, but not on ET-1 receptor expression or Ca²⁺ sensitization. In addition, this aortic hyporeactivity to ET-1 is dependent on protein synthesis. The pulmonary artery is not affected because LPS induces a desensitization of the sGC/cGMP dependent pathway by decreasing protein expression levels of sGCβ₁, and hence sGC activity, and increasing PDE5 activity. Neither the endothelium, cyclooxygenase, reactive oxygen species nor K⁺ channels are involved in these LPS effects. Therefore, it is likely that both Ca²⁺ homeostasis and the sGC/cGMP pathway play important roles in vasomotor complications in sepsis. sGC and/or PDE5-selective inhibitors, together with manipulating VSMC [Ca²⁺]i, could be important in controlling systemic and pulmonary vasomotor complications in sepsis.
Supervisor: Watson, Malcolm ; Smirnov, Sergey Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: calcium ; inflammation ; vascular ; pulmonary ; nitric oxide ; endothelin ; lippolysaccharide