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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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PKA-mediated phosphorylation of cTnI and PLB is not induced by pretreatment with individual isoform-selective PDE inhibitors. Cultured ARVM were exposed to vehicle, IBMX (100 μM), EHNA (10 μM), cilostamide (10 μM), rolipram (10 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. Representative western blots of phosphoSer22/23 cTnI, phosphoSer16 PLB and total PLB, as indicated
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Fig3: PKA-mediated phosphorylation of cTnI and PLB is not induced by pretreatment with individual isoform-selective PDE inhibitors. Cultured ARVM were exposed to vehicle, IBMX (100 μM), EHNA (10 μM), cilostamide (10 μM), rolipram (10 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. Representative western blots of phosphoSer22/23 cTnI, phosphoSer16 PLB and total PLB, as indicated

Mentions: The lack of effect of the isoform-selective PDE inhibitors on ET1-induced PKD activation could be due to their failure to elevate cAMP and activate PKA in the relevant myocyte compartment. To explore whether the isoform-selective PDE inhibitors have a different impact on PKA activity in other myocyte compartments, we also examined their effects on the phosphorylation status of cTnI and PLB. As shown in Fig. 3, whilst non-selective PDE inhibition with IBMX or exposure to ISO markedly increased the phosphorylation of cTnI and PLB at their PKA sites, the isoform-selective PDE inhibitors produced little effect. These findings suggest that in the presence of basal adenylate cyclase activity, inhibition of any individual PDE isoform fails to produce a sufficient increase in PKA activity in the myocyte compartments that facilitate the inhibition of PKD activation or the induction of cTnI or PLB phosphorylation.Fig. 3


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)

PKA-mediated phosphorylation of cTnI and PLB is not induced by pretreatment with individual isoform-selective PDE inhibitors. Cultured ARVM were exposed to vehicle, IBMX (100 μM), EHNA (10 μM), cilostamide (10 μM), rolipram (10 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. Representative western blots of phosphoSer22/23 cTnI, phosphoSer16 PLB and total PLB, as indicated
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Related In: Results  -  Collection

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Fig3: PKA-mediated phosphorylation of cTnI and PLB is not induced by pretreatment with individual isoform-selective PDE inhibitors. Cultured ARVM were exposed to vehicle, IBMX (100 μM), EHNA (10 μM), cilostamide (10 μM), rolipram (10 μM) or ISO (100 nM) for 10 min, followed by vehicle (con) or ET1 (100 nM) for 10 min. Representative western blots of phosphoSer22/23 cTnI, phosphoSer16 PLB and total PLB, as indicated
Mentions: The lack of effect of the isoform-selective PDE inhibitors on ET1-induced PKD activation could be due to their failure to elevate cAMP and activate PKA in the relevant myocyte compartment. To explore whether the isoform-selective PDE inhibitors have a different impact on PKA activity in other myocyte compartments, we also examined their effects on the phosphorylation status of cTnI and PLB. As shown in Fig. 3, whilst non-selective PDE inhibition with IBMX or exposure to ISO markedly increased the phosphorylation of cTnI and PLB at their PKA sites, the isoform-selective PDE inhibitors produced little effect. These findings suggest that in the presence of basal adenylate cyclase activity, inhibition of any individual PDE isoform fails to produce a sufficient increase in PKA activity in the myocyte compartments that facilitate the inhibition of PKD activation or the induction of cTnI or PLB phosphorylation.Fig. 3

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