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No description available.

No description available.

No description available.

L-type Ca2+ channels, found in vascular smooth muscle cells, function to control Ca2+ influx, which directly regulates the degree of tension in the vasculature. An influx of Ca2+ causes these cells to contract while inhibition of this channel causes muscle relaxation, a major goal in treating hypertension. Acute hypoxia inhibits, and chronic hypoxia enhances, Ca2+ channel currents. The mechanisms underlying these hypoxic responses were examined in HEK 293 cells by altering cellular levels of proposed mediators of 0 2 sensing which have previously been shown to be involved in the redox model of 02 sensing in various cell types. In these studies I investigated the roles of mitochondrial complexes and NADPH oxidase function, and changes in cellular ROS levels, on the acute and chronically hypoxic regulation of recombinant L-type Ca2+ channels. An increase in H202, a form of ROS, by exogenous application was found to enhance Ca2+ currents. However neither catalase nor H202 affected the acute hypoxic response. In contrast superoxide dismutase (SOD) abolished hypoxic inhibition of recombinant L-type Ca2+ channels, suggestive of a role of 02- production in 02 sensing. Altered production of this ROS during hypoxia may occur within the mitochondria since acute 02 sensing was abolished in mitochondria-depleted p0 cells. Alterations in NADPH oxidase activity via application of NADPH oxidase inhibitors such as DPl and P AO did not mediate the acute hypoxic response. Hypoxic regulation of mitochondrial complex I may also mediate the response to chronic hypoxia since current enhancement by this stimulus was abolished by rotenone. These findings support the involvement of altered mitochondrial function in the 0 2 sensing pathway which mediates the hypoxic responses of recombinant L-type Ca2+ channel a1c subunits. / Thesis / Master of Science (MSc)

Improving treatment and prevention of pelvic organ prolapse, a disorder affecting up to half of parous women, requires thorough mechanical analysis of the vagina and other endopelvic structures at the cellular level. In this study, we tested single vaginal smooth muscle cells (SMCs) to quantify their elastic moduli. Cells were enzymatically isolated from vaginal walls of freshly sacrificed, virgin Long Evans rats and cultured using well-established methods. A custom-built experimental setup was used to perform tensile tests. Micropipettes were fabricated to serve as cantilever-type load cells, which were coated in cellular adhesive. Two pipettes applied tension to SMCs until adhesion between the cell and a pipette failed. During mechanical testing, images of SMCs were collected and translated into strain and stress. Specifically, force/stress data were calculated using Euler-Bernoulli Beam Theory and by making simplifying geometric assumptions. The average initial and total elastic moduli (mean ± SEM) for single vaginal SMCs were 6.06 ± 0.26 kPa and 5.4 ± 0.24 kPa, respectively, which is within the range reported for other types of SMCs, mainly airway and vascular, of various species. This protocol can and will be applied to further investigate mechanics of single cells from the pelvic region with independent variables such as parity, age, body mass index, and various stages of POP. Results of these experiments will provide critical information for improving current treatments like drug therapies, surgical procedures, medical grafts and implants, and preventative practices like stretching and exercise techniques. / Master of Science

Yes / Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically. / This work was supported by the British Heart Foundation.

From animal experimentation, and studies using in vitro models, there was evidence in the literature to suggest that dietary fibre may influence contractility and motility of the gastrointestinal tract and long chain (LC) n-3 polyunsaturated fatty acids (PUFAs) from marine sources may influence contractility of smooth muscle cells in blood vessels. The hypothesis of this thesis was that dietary fish oil and/or fibre influence the contractility of isolated intact sections of gut smooth muscle tissue from small animal models. Methodology was established to measure in vitro contractility of intact pieces of guinea pig ileum with the serosal side isolated from the lumen. It was demonstrated that four amino acid peptides from κ-casein (casoxins) applied to the lumen overcame morphine-induced inhibition of contraction. Using this established technology, the guinea pig was used to investigate the effects of dietary fibre and fish oil supplementation on gut in vitro contractility. In separate experiments, changes in sensitivity to electrically-driven and 8-iso-prostanglandin (PG)E₂-induced contractility were demonstrated for dietary fibre and fish oil. A modified, isolated gut super-perfusion system was then established for the rat to validate these findings. It was subsequently shown that LC n-3 PUFA from dietary fish oil significantly increased maximal contraction in response to the G-protein coupled receptor modulators, acetylcholine and the eicosanoids PGE₂, PGF₂α, 8-iso-PGE₂ and U-46619 in ileum but not colon, without changes in sensitivity (EC₅₀), when n-3 PUFA as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) had been incorporated to a similar degree into the gut total phospholipid membrane pool. It was further established that the spontaneously hypertensive rat (SHR) had a depressed prostanoid (PGE₂and PGF₂α) response in the gut that could be restored by dietary fish oil supplementation (5% w/w of total diet) in the ileum but not the colon. Importantly, the muscarinic response in the colon of the SHR was increased by fish oil supplementation with DHA likely to be the active agent. Dietary fish oil dose experiments deduced differential increases in response occurred at fish oil concentrations of 1% for muscarinic and 2.5% (w/w) for prostanoid stimulators of the ileum with no difference in receptor-independent KCl-induced depolarization-driven contractility. Studies combining high amylose resistant starch (HAMS, 10% w/w) and fish oil (10% w/w) fed to young rats demonstrated a low prostanoid response that was enhanced by dietary fish oil but not resistant starch. There was however, an interactive effect of the HAMS and fish oil noted for the muscarinic-mimetic, carbachol. Generally, resistant starch increased the large bowel short chain fatty acid pool with a subsequent lower pH. Binding studies determined that while the total muscarinic receptor binding properties of an isolated ileal membrane fraction were not affected in mature rats by dietary fish oil, young rats had a different order of muscarinic receptor subtype response with a rank order potency of M₃ > M₁ > M₂ compared to mature animals of M₃ > M₂ > M₁ with fish oil altering the sensitivity of the M₁ receptor subtype in isolated carbachol-precontracted ileal tissue. In conclusion, experiments using the guinea pig and rat gut models demonstrated that dietary fish oil supplementation, and to a lesser degree fibre, increased receptor-driven contractility in normal and compromised SHR ileum and colon. Further, changes in responsiveness were demonstrated in the developing rat gut prostanoid and muscarinic receptor populations that could be altered by dietary fish oil. Preliminary evidence suggested that fish oil as DHA may alter receptor-driven gut contractility by mechanisms involving smooth muscle calcium modulation. Defining the role that dietary fibre and fish oil, and other nutrients, play in normal and diseased states of bowel health such as inflammatory bowel disease (IBD), where contractility is compromised, are among the ongoing challenges. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1316907 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

From animal experimentation, and studies using in vitro models, there was evidence in the literature to suggest that dietary fibre may influence contractility and motility of the gastrointestinal tract and long chain (LC) n-3 polyunsaturated fatty acids (PUFAs) from marine sources may influence contractility of smooth muscle cells in blood vessels. The hypothesis of this thesis was that dietary fish oil and/or fibre influence the contractility of isolated intact sections of gut smooth muscle tissue from small animal models. Methodology was established to measure in vitro contractility of intact pieces of guinea pig ileum with the serosal side isolated from the lumen. It was demonstrated that four amino acid peptides from κ-casein (casoxins) applied to the lumen overcame morphine-induced inhibition of contraction. Using this established technology, the guinea pig was used to investigate the effects of dietary fibre and fish oil supplementation on gut in vitro contractility. In separate experiments, changes in sensitivity to electrically-driven and 8-iso-prostanglandin (PG)E₂-induced contractility were demonstrated for dietary fibre and fish oil. A modified, isolated gut super-perfusion system was then established for the rat to validate these findings. It was subsequently shown that LC n-3 PUFA from dietary fish oil significantly increased maximal contraction in response to the G-protein coupled receptor modulators, acetylcholine and the eicosanoids PGE₂, PGF₂α, 8-iso-PGE₂ and U-46619 in ileum but not colon, without changes in sensitivity (EC₅₀), when n-3 PUFA as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) had been incorporated to a similar degree into the gut total phospholipid membrane pool. It was further established that the spontaneously hypertensive rat (SHR) had a depressed prostanoid (PGE₂and PGF₂α) response in the gut that could be restored by dietary fish oil supplementation (5% w/w of total diet) in the ileum but not the colon. Importantly, the muscarinic response in the colon of the SHR was increased by fish oil supplementation with DHA likely to be the active agent. Dietary fish oil dose experiments deduced differential increases in response occurred at fish oil concentrations of 1% for muscarinic and 2.5% (w/w) for prostanoid stimulators of the ileum with no difference in receptor-independent KCl-induced depolarization-driven contractility. Studies combining high amylose resistant starch (HAMS, 10% w/w) and fish oil (10% w/w) fed to young rats demonstrated a low prostanoid response that was enhanced by dietary fish oil but not resistant starch. There was however, an interactive effect of the HAMS and fish oil noted for the muscarinic-mimetic, carbachol. Generally, resistant starch increased the large bowel short chain fatty acid pool with a subsequent lower pH. Binding studies determined that while the total muscarinic receptor binding properties of an isolated ileal membrane fraction were not affected in mature rats by dietary fish oil, young rats had a different order of muscarinic receptor subtype response with a rank order potency of M₃ > M₁ > M₂ compared to mature animals of M₃ > M₂ > M₁ with fish oil altering the sensitivity of the M₁ receptor subtype in isolated carbachol-precontracted ileal tissue. In conclusion, experiments using the guinea pig and rat gut models demonstrated that dietary fish oil supplementation, and to a lesser degree fibre, increased receptor-driven contractility in normal and compromised SHR ileum and colon. Further, changes in responsiveness were demonstrated in the developing rat gut prostanoid and muscarinic receptor populations that could be altered by dietary fish oil. Preliminary evidence suggested that fish oil as DHA may alter receptor-driven gut contractility by mechanisms involving smooth muscle calcium modulation. Defining the role that dietary fibre and fish oil, and other nutrients, play in normal and diseased states of bowel health such as inflammatory bowel disease (IBD), where contractility is compromised, are among the ongoing challenges. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1316907 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Background-Previously we found that smooth muscle cell (SMC)-specific knockout of miR-17 similar to 92 attenuates hypoxia-induced pulmonary hypertension. However, the mechanism underlying miR-17 similar to 92-mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR-17 similar to 92 regulates hypoxia-inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs). Methods and Results-We show that hypoxic sm-17 similar to 92(-/-) mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm-17 similar to 92 (-/-) mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR-17 similar to 92 inhibitors suppress hypoxia-induced levels of HIF1 alpha, VEGF, Glut1, HK2, and PDK1 but not HIF2 alpha in vitro in PASMC. Overexpression of miR-17 in PASMC represses PHD2 expression, whereas miR-17/20a inhibitors induce PHD2 expression. The 3'-UTR of PHD2 contains a functional miR-17/20a seed sequence. Silencing of PHD2 induces HIF1a and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1 alpha and cell proliferation. SMC-specific knockout of PHD2 enhances hypoxia-induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients. Conclusions-Our results suggest that PHD2 is a direct target of miR-17/20a and that miR-17 similar to 92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1 alpha.