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

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.