PDE1 inhibition modulates Cav1.2 channel to stimulate cardiomyocyte contraction

ABSTRACTRationaleCyclic adenosine monophosphate (cAMP) activation of protein kinase A (PKA) stimulates excitation-contraction coupling, increasing cardiac contractility. This is clinically leveraged by beta-adrenergic stimulation (β-ARs) or phosphodiesterase-3 inhibition (PDE3i), though both approaches are limited by arrhythmia and chronic myocardial toxicity. Phosphodiesterase-1 inhibition (PDE1i) also augments cAMP and was recently shown in rabbit cardiomyocytes to augment contraction independent of β-AR stimulation or blockade, and with less intracellular calcium rise than β-ARs or PDE3i. Early testing of PDE1 inhibition in humans with neuro-degenerative disease and dilated heart failure has commenced. Yet, the molecular mechanisms for PDE1i inotropic effects remain largely unknown.ObjectiveDefine the mechanism(s) whereby PDE1i increases contractility.Methods and ResultsPrimary guinea pig myocytes which express the cAMP-hydrolyzing PDE1C isoform found in larger mammals and humans were studied. The potent, selective PDE1i (ITI-214) did not alter cell shortening or Ca2+ transients under resting conditions whereas both increased with β-ARs or PDE3i. However, PDE1i enhanced shortening with less Ca2+ rise in a PKA-dependent manner when combined with low-dose adenylate cyclase stimulation (Forskolin). Unlike PDE3i, PDE1i did not augment β-AR responses. Whereas β-ARs reduced myofilament Ca2+ sensitivity and increased sarcoplasmic reticular Ca2+ content in conjunction with greater phosphorylation of troponin I, myosin binding protein C, and phospholamban, PDE1i did none of this. However, PDE1i increased Cav1.2 channel conductance similar to PDE3i in a PKA-dependent manner. Myocyte shortening and peak Ca2+ transients were more sensitive to Cav1.2 blockade with nitrendipine combined with PDE1i versus PDE3i. Lastly, PDE1i was found to be far less arrythmogenic than PDE3i.ConclusionsPDE1i enhances contractility by a PKA-dependent increase in Cav1.2 conductance without concomitant myofilament desensitization. The result is less rise in intracellular Ca2+ and arrhythmia compared to β-ARs and/or PDE3i. PDE1i could be a novel positive inotrope for failing hearts without the toxicities of β-ARs and PDE3i.

cAMP-Mediated and Metabolic Amplification of Insulin Secretion Are Distinct Pathways Sharing Independence of β-Cell Microfilaments

Insulin secretion is triggered by an increase in the cytosolic calcium concentration ([Ca2+]c) in β-cells. Ca2+-induced exocytosis of insulin granules can be augmented by metabolic amplification (unknown signals generated through glucose metabolism) or neurohormonal amplification (in particular cAMP mediated). Functional actin microfilaments are not required for metabolic amplification, but their possible role in cAMP-mediated amplification is unknown. It is also uncertain whether cAMP (generated in response to glucose) is implicated in metabolic amplification. These questions were addressed using isolated mouse islets. cAMP levels were increased by phosphodiesterase inhibition (with isobutylmethylxanthine) and adenylate-cyclase stimulation (with forskolin or glucagon-like peptide-1, 7-36 amide). Raising cAMP levels had no steady-state impact on actin polymerization in control islets. Neither disruption (depolymerization by latrunculin) nor stabilization (polymerization by jasplakinolide) of actin microfilaments was counteracted by cAMP. Both changes increased both phases of glucose- or tolbutamide-induced insulin secretion but did not prevent further amplification by cAMP. These large changes in secretion were not caused by changes in [Ca2+]c, which was only slightly increased by cAMP. Both phases of insulin secretion were larger in response to glucose than tolbutamide, although [Ca2+]c was lower. This difference in secretion, which reflects metabolic amplification, was independent of microfilaments, was not attributable to differences in cAMP, and persisted in presence of dibutyryl-cAMP or when cAMP levels were variably raised by isobutylmethylxanthine + forskolin or glucagon-like peptide-1, 7-36 amide. We conclude that metabolic and cAMP-mediated amplification of insulin secretion are distinct pathways that accelerate acquisition of release competence by insulin granules that can access exocytotic sites without intervention of microfilaments.

Agonist regulation of adenylate cyclase activity in neuroblastoma × glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the β2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity

Stable cell lines, derived from NG108-15 cells and transfected to express both the β2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the β2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5´-[β,γ-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced β2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration–effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones overexpressing adenylate cyclase type II. A maximally effective concentration of the β-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5´-N-ethylcarboxamidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

Identification of a receptor for ADP on blood platelets by photoaffinity labelling

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5′-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase.

Hepatic changes during a carrageenan induced granuloma in rats

Hepatic changes during inflammation were studied in rats bearing a carrageenan induced granuloma. In spite of a decrease in the metabolic capacity of microsomes to induce lipid peroxidation during inflammation, the endogenous lipid peroxidation remained unchanged and unrelated with the hepatic activities measured. The continuous increase in hepatic cAMP observed during acute and chronic phases could be related to adenylate cyclase stimulation by mediators, and could be an initial step in the hepatocyte adaptation leading to the increased level of hepatic caeruloplasmin, to the reduction of cytochrome P-450 level and to the modifications of Ca2+sequestration by microsomes.