• Activation of signal transduction mechanisms by angiotensin II in cultured cardiac myocytes

      Lokuta, Andrew Joseph; Rogers, Terry Birkby (1993)
      Angiotensin II (AngII), a potent peptide hormone, has a well-characterized acute effect on vascular smooth muscle. Recently however, an interest has developed concerning both short term and long term AngII-mediated effects in cardiac muscle cells. Presently, little is known about the biochemical signaling mechanisms activated in cardiac muscle by AngII. To address this issue, experiments were designed using cultured neonatal rat ventricular myocytes exposed to AngII, and then analyzed with biochemical assays to examine specific molecular effects. As a first step, existing procedures for culturing neonatal rat cardiocytes had to be improved. The goal was to further purify otherwise heterogeneous cultures of dissociated ventricular cells into homogeneous cultures of spontaneously contracting cardiocytes. After several established methods were investigated, the use of {dollar}\gamma{dollar}-irradiation at low doses (3500 rads total dose), 24 hours after initially plating the cells in plastic 1.7 cm culture wells, was developed and thoroughly characterized for use in our culture system. Further methods were developed to help maintain these cultures in the absence of serum for 10 to 14 days. The next major goal was to examine signaling pathways which AngII might stimulate in these serum-free, spontaneously contracting cardiocytes. With the use of ({dollar}\sp3{dollar}H) arachidonic acid and ({dollar}\sp3{dollar}H) inositol to label membrane phospholipids, significant evidence was gathered that showed AngII activates two different phospholipases thus generating multiple phospholipid-derived second messengers. Thin layer chromatography, gas chromatography-mass spectrometry, and enzyme inhibition assays revealed that both phospholipase C and phospholipase A{dollar}\sb2{dollar} were activated by AngII in these cardiac cells. Their activation leads to the generation of several second messengers including inositol polyphosphates, diacylglycerol, and unesterified arachidonic acid. Further, receptor subtype-specific antagonists revealed that these processes are mediated in part by two different AngII receptor subtypes, the nonpeptide Losartan-sensitive AT{dollar}\sb1{dollar} and the peptide CGP 42112A-sensitive AT{dollar}\sb2.{dollar} These results indicate that cultured neonatal rat ventricular myocytes, like many other types of cells, are subject to regulation by AngII. A specific role for the second messenger molecules released following exposure to AngII, or the reason why AngII stimulates multiple signaling pathways in a seemingly coordinated fashion through subtype-specific receptors, remains to be elucidated.
    • Biochemical signalling responses of cultured neonatal rat heart myocytes to angiotensin II

      Abdellatif, Maha M.; Rogers, Terry Birkby (1991)
      Angiotensin II (AngII) has previously been shown to increase the beating frequency, decrease the force of contraction and induce phosphoinositide hydrolysis in cultured neonatal heart myocytes. These responses to AngII were transient, after which the cells became insensitive to any further application of the hormone. When the myocytes were incubated with 100 nM AngII there was a marked increase in total inositol phosphate (IPs) levels, that was maximal after 1 min, and returned to basal values after 5 min. This rapid desensitization process was concentration independent, and was completely reversible after 1 hr of hormone removal. The phenomenon was not due to an increase in the rate of degradation of inositol phosphates or depletion of the phospholipid pools. The homologous nature of the AngII-evoked desensitization, provided evidence that the receptor was the target of modification in this process. Binding studies with ({dollar}\sp{lcub}125{rcub}{dollar}I) AngII, revealed that the loss of surface receptors was not responsible for desensitization. Although activation of protein kinase C (PKC), desensitized the cells to AngII a role for the kinase in the hormone-evoked desensitization is not likely since the phenomenon was still observed in PKC-down regulated. The initial rapid burst of AngII evoked phosphoinositide turnover, was followed by a sustained (3 hr) low level of turnover. It was hypothesized that this long term signal may mediate a growth effect of the hormone on the heart cells. Consistent with this view, it was found that AngII increased the level of c-fos mRNA maximally after 0.5 hr. This response to the hormone was dose-dependent and mediated by the subtype-1 receptor through both PKC-dependent and independent pathways, but not through the initial 30s burst of inositol phosphates accumulation. The PKC-independent pathway was also independent of extracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar}, arachidonic acid metabolites, calmodulin and the N{dollar}\sp{lcub}+{rcub}{dollar}/H{dollar}\sp{lcub}+{rcub}{dollar} exchanger. In conclusion, AngII induces a short lived, rapid hydrolysis of phosphoinositides, followed by a slower sustained effect in neonatal rat cardiac myocytes. The mechanism of desensitization of the cells to the former effect of the hormone is at the level of the receptor, but is independent of loss of surface receptors of PKC activation. (Abstract shortened with permission of author.)
    • Characterization of angiotensin II and protein kinase C signalling pathways that regulate intracellular pH in neonatal rat ventricular myocytes

      Kohout, Trudy Ana; Rogers, Terry Birkby (1995)
      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.
    • Novel Functions of Protein Kinase D in Cardiac Excitation-Contraction Coupling

      Goodall, Mariah; Rogers, Terry Birkby (2010)
      While the function of protein kinase D (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 and CREB implicating this kinase in the development of dysfunction seen in heart failure. Here we significantly extend our understanding of PKD signaling in heart using a molecular genetic approach to examine PKD through adenoviral vector expression of wild type (wt), constitutively active (ca) or dominant negative (dn) PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated-reticular pattern suggesting the importance of PKD in Ca<super>2+</super> signaling in heart. Thus, an initial hypothesis was that PKD plays unappreciated roles in regulation of excitation-contraction coupling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca<super>2+</super> sensitivity with an unexpected electrical stimulus-dependence to this response. This desensitization was accompanied by stimulus-dependent increases in phosphorylation of cTnI and of regulatory site, Ser916, on PKD. The functional importance of this phospho-Ser916 event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. dnPKD expression resulted in sensitization of the myofilaments to Ca<super>2+</super> and blocked stimulus-dependent increases in cTnI phosphorylation. Furthermore, steady-state Ca<super>2+</super> transients were markedly increased in dnPKD cells and are explained, in part, by a marked increase in sarcoplasmic reticulum (SR) Ca<super>2+</super> load. In addition, changes in the cardiac Ca<super>2+</super> current (I<sub>Ca</sub>) and behavior of the phosphatase inhibitor, calyculin A (CalyA), support a role for PKD as a dynamic regulatory kinase of the L-type Ca<super>2+</super> channel (LTCC). Together, these data suggest a complex collection of novel functions carried out by PKD to dynamically regulate several components of the excitation-contraction coupling cascade in cardiomyocytes to allow for precise fine-tuning of cardiac cell function. Given that PKD activity is elevated in failing hearts, it will be important to determine the role of increased signaling at these newly appreciated cellular locales in the development of heart disease.