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Regulation by phosphodiesterase isoforms of protein kinase A-mediated attenuation of myocardial protein kinase D activation.

Haworth RS, Cuello F, Avkiran M - Basic Res. Cardiol. (2010)

Bottom Line: However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation.Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective.Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.

View Article: PubMed Central - PubMed

Affiliation: King's College London British Heart Foundation Centre, Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London, SE1 7EH, UK. robert.haworth@kcl.ac.uk

ABSTRACT
Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.

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ET1-induced PKD activation is inhibited by pretreatment with IBMX or isoprenaline. Cultured ARVM were exposed to vehicle, IBMX (100 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. a Representative western blots of phosphoSer916 PKD, phosphoSer744/748 PKD, phosphoSer498 HDAC5 and total PKD, as indicated. b Quantitative data for the ET1-induced change in phosphoSer916 PKD, phosphoSer744/748 PKD, and the upper and lower bands from the pSer498 HDAC5 blots (n = 4). *P < 0.05 versus vehicle control
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Fig1: ET1-induced PKD activation is inhibited by pretreatment with IBMX or isoprenaline. Cultured ARVM were exposed to vehicle, IBMX (100 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. a Representative western blots of phosphoSer916 PKD, phosphoSer744/748 PKD, phosphoSer498 HDAC5 and total PKD, as indicated. b Quantitative data for the ET1-induced change in phosphoSer916 PKD, phosphoSer744/748 PKD, and the upper and lower bands from the pSer498 HDAC5 blots (n = 4). *P < 0.05 versus vehicle control

Mentions: We first determined whether non-selective PDE inhibition inhibits ET1-induced PKD activation in ARVM. The phosphorylation status of heterologously expressed PKD at Ser916 (autophosphorylation) and Ser744/748 (activation loop phosphorylation by PKC) was determined by western blotting. As illustrated in Fig. 1a and shown quantitatively in Fig. 1b, exposure of ARVM to ET1 (100 nM for 10 min) promoted robust phosphorylation of PKD at both the activation loop sites and the autophosphorylation site, reflecting PKC-dependent activation of PKD. Consistent with our previous work [17], pretreatment with the β-AR agonist ISO (100 nM for 10 min) substantially reduced ET1-induced PKD phosphorylation. Pretreatment with the non-selective PDE inhibitor IBMX (100 μM for 10 min) also reduced ET1-induced PKD phosphorylation at both the Ser744/748 and the Ser916 sites to a similar level to that achieved with ISO pretreatment. This suggests that global PDE inhibition in the presence of basal adenylate cyclase activity produces a sufficient increase in PKA activity in the relevant compartment to inhibit PKD activation.Fig. 1


Regulation by phosphodiesterase isoforms of protein kinase A-mediated attenuation of myocardial protein kinase D activation.

Haworth RS, Cuello F, Avkiran M - Basic Res. Cardiol. (2010)

ET1-induced PKD activation is inhibited by pretreatment with IBMX or isoprenaline. Cultured ARVM were exposed to vehicle, IBMX (100 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. a Representative western blots of phosphoSer916 PKD, phosphoSer744/748 PKD, phosphoSer498 HDAC5 and total PKD, as indicated. b Quantitative data for the ET1-induced change in phosphoSer916 PKD, phosphoSer744/748 PKD, and the upper and lower bands from the pSer498 HDAC5 blots (n = 4). *P < 0.05 versus vehicle control
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Related In: Results  -  Collection

Show All Figures
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Fig1: ET1-induced PKD activation is inhibited by pretreatment with IBMX or isoprenaline. Cultured ARVM were exposed to vehicle, IBMX (100 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. a Representative western blots of phosphoSer916 PKD, phosphoSer744/748 PKD, phosphoSer498 HDAC5 and total PKD, as indicated. b Quantitative data for the ET1-induced change in phosphoSer916 PKD, phosphoSer744/748 PKD, and the upper and lower bands from the pSer498 HDAC5 blots (n = 4). *P < 0.05 versus vehicle control
Mentions: We first determined whether non-selective PDE inhibition inhibits ET1-induced PKD activation in ARVM. The phosphorylation status of heterologously expressed PKD at Ser916 (autophosphorylation) and Ser744/748 (activation loop phosphorylation by PKC) was determined by western blotting. As illustrated in Fig. 1a and shown quantitatively in Fig. 1b, exposure of ARVM to ET1 (100 nM for 10 min) promoted robust phosphorylation of PKD at both the activation loop sites and the autophosphorylation site, reflecting PKC-dependent activation of PKD. Consistent with our previous work [17], pretreatment with the β-AR agonist ISO (100 nM for 10 min) substantially reduced ET1-induced PKD phosphorylation. Pretreatment with the non-selective PDE inhibitor IBMX (100 μM for 10 min) also reduced ET1-induced PKD phosphorylation at both the Ser744/748 and the Ser916 sites to a similar level to that achieved with ISO pretreatment. This suggests that global PDE inhibition in the presence of basal adenylate cyclase activity produces a sufficient increase in PKA activity in the relevant compartment to inhibit PKD activation.Fig. 1

Bottom Line: However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation.Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective.Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.

View Article: PubMed Central - PubMed

Affiliation: King's College London British Heart Foundation Centre, Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London, SE1 7EH, UK. robert.haworth@kcl.ac.uk

ABSTRACT
Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.

Show MeSH
Related in: MedlinePlus