Browsing by Author "Macdonald, Justin Anthony"

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    Novel Regulators of Vascular Myogenic Tone: A Focus on SMTNL1 and ZIPK
    (2018-09-19) Turner, Sara Rose; Macdonald, Justin Anthony; Walsh, Michael P.; Cole, William C.; Slater, Donna M.; Thompson, Jennifer; Nixon, Graeme F.
    Myogenic constriction in the resistance vasculature plays a fundamental role in the regulation of blood flow, maintenance of mean arterial pressure and in promoting overall cardiovascular health. Myogenic control of these arteries is an innate function of the vascular smooth muscle and is activated by pressure-dependent mechanisms of Ca2+-CaM-MLCK activation, Ca2+ sensitization and cytoskeletal reorganization. Recently, two proteins of interest to our research group were found to contribute to Ca2+ sensitization in vascular smooth muscle, smoothelin-like 1(SMTNL1) and zipper-interacting protein kinase (ZIPK). SMTNL1 is a relatively unknown protein which may act as an inhibitor of MLCP. ZIPK is a Ser/Thr kinase capable of phosphorylating LC20, MYPT1 and CPI-17 proteins among other targets. Neither SMTNL1 nor ZIPK has been previously investigated for a role in contributing to the regulation of the vascular myogenic response, and this investigation forms the core of this thesis. The findings presented here identify: (1) enhanced myogenic response in the mesenteric arteries of the male SMTNL1 KO mouse corresponding with (2) significant potential for Ca2+ sensitization via down regulation of MYPT1 and upregulation of CPI-17, and (3) the first evidence for ZIPK contribution to the vascular myogenic response of the rat cerebral and cremasteric arteries. While these findings were limited to the healthy vasculature, common chronic diseases such as hypertension and type 2 diabetes mellitus are known to be associated with pathological alterations in the myogenic response of the vasculature. Using a novel and specific ZIPK inhibitor, HS38, we identified a role for ZIPK activity in mediating some of the maladaptations of the vasculature in rodent models of hypertension (the SHR) and type 2 diabetes (the GK rat). In summary, these findings suggest both SMTNL1 and ZIPK play important roles in the regulation of healthy vascular function and may provide new avenues for exploration into the dysfunction of the vasculature in pathological conditions.
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    The Spatial-temporal Contribution of Prostaglandin E2 Pathway to Rat Myometrial Contractile Function during Pregnancy and Labour
    (2020-09-24) Liwa, Anthony Cuthbert; Slater, Donna M.; Cole, William C.; Davidge, Sandra Thomas; Macdonald, Justin Anthony; Giembycz, Mark A.; Roth, Sheldon H.
    A key function of the uterus during pregnancy is to accommodate the growing fetus in a relatively quiescent environment. Towards the end of pregnancy, the uterus transforms to an active organ capable of generating forceful contractions during labour. Prostaglandins are thought to play a key role in the parturition process, with prostaglandin E2 (PGE2) being produced by uterine tissues, and having possible roles including stimulation of cervical ripening and uterine contractions. PGE2 exhibits diverse physiological actions, most probably depending on expression levels of selective PGE2 (EP) receptor subtypes; in the uterus EP1 and EP3 receptors may stimulate myometrial contraction, whereas EP2 and EP4 receptors may evoke relaxation. There is no consensus concerning their relative importance in regulating uterine function, and whether there are spatial- and/or time-dependent alterations in how PGE2 regulates uterine contractility via changes in PGE2 synthesis and/or EP receptor expression. We have hypothesised that, in myometrial tissue, expression of enzymes involved in PGE2 synthesis and/or the four EP receptors are regulated to provide for region- and time-dependent variations in the contribution of PGE2 to uterine quiescence in pregnancy and contractility in labour. An in vitro tissue bath was utilised to examine myometrial contractility responses to PGE2 and EP antagonists. Real-time polymerase chain reaction and western blotting experiments were used to determine expression of key enzymes involved in PGE2 synthesis plus the EP receptors. Uterine tissues from upper and lower uterine tissues at days 15-21 of gestation and during labour were used for the experiments. Our findings demonstrate the presence of spontaneous contractions in ex vivo uterine tissues at all time points studied. No regional difference in the magnitude of uterine contraction was observed with the exception of upper day 21, in which contractility was greater than in the upper compared to the lower uterus. The addition of PGE2 increased the spontaneous contractions in non-pregnant and day 15-21 pregnant tissues, but not those from rats in active labour. EP3 antagonist (L-798106) did not alter the actions of PGE2, whereas PGE2-evoked contractions of day 18 pregnant, but not in labour myometrium, were enhanced in the presence of EP4 antagonist (ONO-AE3-208). No significant differences in the expression of PGE2 isoenzyme and EP receptor mRNA and protein expression were detected in upper or lower uterine tissues during pregnancy and labour, with the exception of increased COX1 and decreased EP3 mRNA levels in tissues from rats in labour. These results suggest that rat uterine tissues can synthesize PGE2 throughout pregnancy and parturition but based on assessment of its effect on contractility in vitro, PGE2 only stimulates myometrial contractility during gestation and not in labour. PGE2 may therefore play a role in enhancing contractility of non-labour myometrium, but perhaps not in the regulation of myometrial contractility during labour. Alterations in the expression of PGE2 synthesizing enzymes and EP receptors would not appear to be the primary determinant of uterine tissue responsiveness. Furthermore, our results do not support the concept of functional regionality in terms of response to PGE2 of the rat uterus. The exact mechanisms of PGE2 regulation of uterine contractility await further investigation.