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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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PDE inhibition regulates ET1-induced PKD activity. Cultured ARVM were exposed to vehicle, IBMX (100 μM), cilostamide (10 μM) and rolipram (10 μM), or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. PKD was immunoprecipitated from cell lysates and PKD activity was assessed by in vitro kinase assays using cTnI as substrate. a Representative autoradiogram of phosphorylated cTnI. b Quantitative data for the ET1-induced change in cTnI phosphorylation (n = 3). *P < 0.05 versus vehicle control
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Fig7: PDE inhibition regulates ET1-induced PKD activity. Cultured ARVM were exposed to vehicle, IBMX (100 μM), cilostamide (10 μM) and rolipram (10 μM), or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. PKD was immunoprecipitated from cell lysates and PKD activity was assessed by in vitro kinase assays using cTnI as substrate. a Representative autoradiogram of phosphorylated cTnI. b Quantitative data for the ET1-induced change in cTnI phosphorylation (n = 3). *P < 0.05 versus vehicle control

Mentions: To confirm that the phosphorylation status of Ser916 faithfully reflects the activation status of PKD, we also determined PKD activity by in vitro kinase assays using recombinant cTnI as a substrate. As shown in Fig. 7, exposure of cells to ET1 promoted robust phosphorylation of cTnI by immunoprecipitated PKD in these assays, and this effect was completely blocked by pretreatment of cells with ISO, as we have reported before [17]. Pretreatment with IBMX or the combination of cilostamide and rolipram also substantially reduced cTnI phosphorylation in these assays (Fig. 7), essentially reflecting the changes observed in PKD phosphorylation at Ser916 and Ser744/748 in earlier experiments (Figs. 1, 6). These findings confirm that in this preparation, PKD phosphorylation status at Ser916 is a robust index of PKD activity.Fig. 7


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)

PDE inhibition regulates ET1-induced PKD activity. Cultured ARVM were exposed to vehicle, IBMX (100 μM), cilostamide (10 μM) and rolipram (10 μM), or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. PKD was immunoprecipitated from cell lysates and PKD activity was assessed by in vitro kinase assays using cTnI as substrate. a Representative autoradiogram of phosphorylated cTnI. b Quantitative data for the ET1-induced change in cTnI phosphorylation (n = 3). *P < 0.05 versus vehicle control
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Fig7: PDE inhibition regulates ET1-induced PKD activity. Cultured ARVM were exposed to vehicle, IBMX (100 μM), cilostamide (10 μM) and rolipram (10 μM), or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. PKD was immunoprecipitated from cell lysates and PKD activity was assessed by in vitro kinase assays using cTnI as substrate. a Representative autoradiogram of phosphorylated cTnI. b Quantitative data for the ET1-induced change in cTnI phosphorylation (n = 3). *P < 0.05 versus vehicle control
Mentions: To confirm that the phosphorylation status of Ser916 faithfully reflects the activation status of PKD, we also determined PKD activity by in vitro kinase assays using recombinant cTnI as a substrate. As shown in Fig. 7, exposure of cells to ET1 promoted robust phosphorylation of cTnI by immunoprecipitated PKD in these assays, and this effect was completely blocked by pretreatment of cells with ISO, as we have reported before [17]. Pretreatment with IBMX or the combination of cilostamide and rolipram also substantially reduced cTnI phosphorylation in these assays (Fig. 7), essentially reflecting the changes observed in PKD phosphorylation at Ser916 and Ser744/748 in earlier experiments (Figs. 1, 6). These findings confirm that in this preparation, PKD phosphorylation status at Ser916 is a robust index of PKD activity.Fig. 7

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