Browsing by Author "Cole, William C."

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    Comparative and functional analysis of β-adrenoceptors in human myometrium
    (2018-01) Albadri, Randa G. A.; Giembycz, Mark A; Slater, Donna M.; Cole, William C.; Klein, Claudia; von der Weid, Pierre-Yves
    There are three subtypes of the β-adrenoceptors (β1, β2 and β3). All the β-adrenoceptor subtypes couple to Gαs subunit of G protein. All the β-adrenoceptor subtypes couple mainly to Gαs subunit of G protein. β2-adrenoceptor agonists have been used clinically to suppress myometrial contractions during the management of preterm labour. β2-adrenoceptor agonists lose their effectiveness in a short time and their use causes several side effects. Evidence in the literature supports the expression of functional β2- and β3-adrenoceptors in the lower pregnant human myometrial tissues. However, no definitive understanding in terms of regional and temporal expression and function of the β-adrenoceptors in the uterus was obtained by reviewing the literature. Experiments of this work have confirmed the expression of the three β-adrenoceptor subtypes in term pregnant (upper and lower) human myometrial tissues by real-time RT-PCR analysis. Poor antibody specificity was an obstacle to determine and localize the β-adrenoceptor proteins in the myometrium by immunohistochemistry. β2- and β3-adrenoceptor agonists did not affect the expression of CRISPLD2, RGS2 and NA4A3 in primary MSM cells after 2 and 6 h of incubation. Further research must be done to determine and localize the expression of the β-adrenoceptors in the myometrium.
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    Development of vascular regulation in the zebrafish embryo.
    (2019-09-19) Bahrami, Nabila; Childs, Sarah J.; Cole, William C.; Gordon, Grant Robert J.
    The vascular system is placed under enormous stress at the onset of cardiac contractility and blood flow. Nascent blood vessel tubes initially consist of a thin endothelial wall and rapidly acquire support from mural cells (pericytes and vascular smooth muscle cells; vSMCs). Following their association with vessels, mural cells acquire vasoactive ability (contraction and relaxation). However, we have little information as to when this vasoactivity first develops, and the extent to which each mural cell type contributes to vascular tone regulation during development. For the first time in an in vivo system, we highlight the dynamic changes in mural cell vasoactivity during development. We assess mural cell vasoactivity in the early zebrafish (Danio rerio) cerebral vasculature in response to pharmacological agents. We determine that pericyte-covered vessels constrict and dilate at 4 days post fertilization (dpf) but not at 6 dpf. The prostaglandin EP4 receptor contributes to pericyte-covered vessel dilation at 4 dpf. In contrast, vSMC-covered vessels constrict but do not dilate at 4 dpf. At 6 dpf, vSMC-covered vessels continue to constrict but only dilate from a pre-constricted state. Using genetic ablation, we demonstrate that mural cell contraction and relaxation is an active response by pericytes and vSMCs. Thus, we show that both pericytes and vSMCs have the ability to regulate cerebral vascular tone but at different stages of development. Pericytes are involved in regulating vessel diameters prior to the maturation of the vSMCs. Once vSMCs mature, pericytes are no longer active, and only vSMCs mediate vasomotor activity in the developing embryonic brain of zebrafish. The onset of vSMC vasoactivity corresponds to the development of increased neuronal activity and neurovascular coupling.
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    Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449
    (Journal of Biological Chemistry, 2009-04-22) Johnson, Rosalyn P.; El-Yazbi, Ahmed F.; Hughes, Morgan F.; Schriemer, David C.; Walsh, Emma J.; Walsh, Michael P.; Cole, William C.
    Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.
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    Investigation of Hydrogen Peroxide/Reactive Oxygen Species-Related Signaling on Vasoactive Responses in Myogenic Resistance Arteries
    (2019-03-28) Kendrick, Dylan John; Braun, Andrew P.; Cole, William C.; Slater, Donna M.; Von Der Weid, Pierre Yves
    The study focuses on the investigation of H2O2 and reactive oxygen species as a putative contributor to endothelium-derived hyperpolarization in myogenically-active resistance arteries, and its contribution to the responses evoked by established vasoactive agents. In particular, I hypothesize that H2O2 and/or ROS serve as physiologic, vasoactive agents in myogenically-active resistance arteries in normal tissue and/or in arteries exhibiting endothelial dysfunction (i.e. conditions with reduced NO bioavailability). Using a number of different experimental protocols such as lucigenin molecular assays provided the amount of NADPH-oxidase inhibition in the presence of apocynin, ML171 and VAS2870, where pressure myography experiments showed the response of rat cremaster arteries to external hydrogen peroxide, indicating a role for hydrogen peroxide within the vasculature. Pressure myography experiments also showed the arterial response to the different NADPH-oxidase inhibitors in terms of baseline myogenic tone. Application of apocynin (10µM and 100µM) further constricted the vessels, ML171 resulted in a transient relaxation, with a full relaxation seen with exposure to higher concentrations (0.1µM, 0.3µM, 10µM) of VAS2870. Responses to established vasoactive agents were observed in the presence of the NADPH-oxidase inhibitors, showing reduced responses at ~50% NADPH-oxidase inhibition, with enhanced responses at greater NADPH-oxidase inhibition. The study shows that the three structurally diverse NADPH-oxidase inhibitors differentially affect basal myogenic tone at concentrations (~IC50 values) that produce comparable inhibition of vascular NADPH-oxidase activity.
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    Mechanisms of rat basilar artery relaxation in health and in a model of subarachnoid hemorrhage
    (2004) Iftinca, Mircea Constantin; Cole, William C.
    Cerebral vasospasm is a primary complication after aneurysmal subarachnoid hemorrhage. Despite a great deal of research regarding the pathogenesis of arterial vasospasm, the mechanisms involved still remain poorly understood and, as a result, the treatment strategies employed today have controversial results in the prevention and treatment of this disease. Within the cerebral circulation, the rate of total cerebral blood flow is held within a relatively narrow range, in spite of wide variations of systemic blood pressure. Among the local regulatory mechanisms, a great deal of interest has been focused on the vascular endothelium and potassium channels, with both believed to play a major role in the regulation of vascular tone. Evidence indicates that the abnormalities in cerebro­vascular function after subarachnoid hemorrhage, in vivo, may involve alterations m endothelial and/or potassium channel function. The present study was undertaken to examine endothelium-dependent and potassium-induced relaxation of rat basilar artery, as well as the involvement of potassium channels in the relaxation mechanisms, under control conditions and in a single injection rat model of subarachnoid hemorrhage. Effects of vasoactive agonists and potassium channel modulators on basilar artery tone were examined using a wire myograph. This study provides evidence for a decrease in endothelial-dependent relaxation in spite of an apparent increase in eNOS protein expression after subarachnoid hemorrhage in the rat single injection model. We found that nitric oxide synthesis/release in the endothelium is dependent on endothelial membrane potential and this mechanism appears to be altered after subarachnoid hemorrhage. We also demonstrated for the first time that the ability of extracellular potassium to induce relaxation of basilar artery is partly dependent on nitric oxide release from the endothelium. In addition, this study demonstrates an increase in ouabain sensitivity of the tissue as well as a possible decrease in inward rectifier potassium channel expression/function after subarachnoid hemorrhage. Pretreatment with cerebro-spinal fluid from subarachnoid hemorrhage, but not control rats depressed acetylcholine- and authentic nitric oxide-induced relaxation of basilar arteries. Treatment with cerebro-spinal fluid obtained from control rats and rats with subarachnoid hemorrhage induced rapid changes in ouabain sensitivity of the tissue.
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    Molecular mechanisms controlling vascular smooth muscle contractility
    (2008) Johnson, Rosalyn; Cole, William C.; Walsh, Michael P.
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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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    Regulation of cerebral arterial myogenic tone by Kv1 channels
    (2006) Chen, Tim; Cole, William C.; Walsh, Michael P.
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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.