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    Characterization of angiotensin II and protein kinase C signalling pathways that regulate intracellular pH in neonatal rat ventricular myocytes

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    Author
    Kohout, Trudy Ana
    Advisor
    Rogers, Terry Birkby
    Date
    1995
    Type
    dissertation
    
    Metadata
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    Abstract
    Angiotensin II (AngII) exerts many functional effects on the heart through the activation of protein kinase C (PKC) to affect contractility, and growth. It is now known that PKC is a family of 11 isoforms designated {dollar}\alpha{dollar}, {dollar}\beta{dollar}I, {dollar}\beta{dollar}II, {dollar}\gamma{dollar}, {dollar}\delta{dollar}, {dollar}\epsilon{dollar}, {dollar}\xi{dollar}, {dollar}\eta{dollar}, {dollar}\theta{dollar}, {dollar}\lambda{dollar}, and {dollar}\mu{dollar}. To examine the effects of PKC on the heart, it was first necessary to characterize which isoforms are expressed in this tissue. A RT-PCR approach was developed to identify isoforms that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Cardiac cDNA was RT-PCR amplified and the products analyzed by a combination of restriction mapping and DNA sequencing which revealed the presence of only the {dollar}\alpha{dollar}, {dollar}\delta{dollar}, {dollar}\epsilon{dollar}, {dollar}\eta{dollar}, and {dollar}\xi{dollar} isoforms cardiac myocytes. Since many cardioactive hormones modulate intracellular pH (pH{dollar}\sb{lcub}\rm i{rcub}{dollar}), the goal of this study was to determine if AngII and PKC altered pH{dollar}\sb{lcub}\rm i{rcub}{dollar} in cultured neonatal rat ventricular myocytes. pH{dollar}\sb{lcub}\rm i{rcub}{dollar} was monitored in single cells loaded with the fluorescent indicator c-SNARF-1 or BCECF. Superfusion with 100 nM TPA, a direct activator of PKC, induces an alkalinization of 0.06 {dollar}\pm{dollar} 0.01 pH unit and increased the initial rate of recovery from an imposed acid load by 2.20 {dollar}\pm{dollar} 0.36 fold. The alkalinization and transporter activation are HCO{dollar}\sb3\sp-{dollar}-independent and amiloride-sensitive indicating the involvement of the Na{dollar}\sp+{dollar}/H{dollar}\sp+{dollar} exchanger. Furthermore, Cl{dollar}\sp-{dollar} removal experiments revealed a TPA-stimulated 1.31 {dollar}\pm{dollar} 0.11 fold enhancement of the acid-loading HCO{dollar}\sb3\sp-{dollar}-/Cl{dollar}\sp-{dollar} exchanger. The increase in the Na{dollar}\sp+{dollar}/H{dollar}\sp+{dollar} activity compared to that of the HCO{dollar}\sb3\sp-{dollar}/Cl{dollar}\sp-{dollar} exchanger is consistent with the alkalinization observed. Stimulation of the myocytes with 100 nM AngII resulted in a rapid HCO{dollar}\sb3\sp-{dollar}-dependent, amiloride-insensitive alkalinization of 0.08 {dollar}\pm{dollar} 0.02 pH unit. AngII also increased the rate of acid extrusion by 3.67 {dollar}\pm{dollar} 0.50 fold in a HCO{dollar}\sb3\sp-{dollar}-dependent and Cl{dollar}\sp-{dollar}-independent manner, indicating the activation of the Na{dollar}\sp+{dollar}/HCO{dollar}\sb3\sp-{dollar}-symport. The AngII activation of the symport is mediated through an AT{dollar}\sb2{dollar}-like signalling pathway since the pH{dollar}\sb{lcub}\rm i{rcub}{dollar} response was blocked by the AT{dollar}\sb2{dollar} receptor antagonist, CGP 42112A, and was unaffected by the AT{dollar}\sb1{dollar} inactivator, DTT. Superfusion of the myocytes with 5 {dollar}\mu{dollar}M arachidonic acid (ARA) mimicked the AngII-mediated alkalinization, suggesting further that ARA may mediate the response. Moreover, the AngII- and the ARA-induced responses were blocked with staurosporine, a PKC inhibitor. In summary, AngII activates the Na{dollar}\sp+{dollar}/HCO{dollar}\sb3\sp-{dollar} symport through the AT{dollar}\sb2{dollar} pathway via ARA and possibly through PKC. Although TPA and AngII both alkalinize the cell, they do so through two distinct pathways, perhaps by activating different PKC isoforms.
    Description
    University of Maryland, Baltimore. Molecular Medicine. Ph.D. 1995
    Keyword
    Biology, Molecular
    Biology, Cell
    Biology, Animal Physiology
    Angiotensin II
    Protein Kinase C
    Myocytes, Cardiac
    Rats
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1553
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    Theses and Dissertations School of Medicine

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