Direct Inhibition of Type 5 Adenylyl Cyclase Prevents Myocardial Apoptosis without Functional Deterioration

Abstract

Adenylyl cyclase, a major target enzyme of ␤-adrenergic receptor signals, is potently and directly inhibited by P-site inhibitors, classic inhibitors of this enzyme, when the enzyme catalytic activity is high. Unlike ␤-adrenergic receptor antagonists, this is a non-or uncompetitive inhibition with respect to ATP. We have examined whether we can utilize this enzymatic property to regulate the effects of ␤-adrenergic receptor stimulation differentially. After screening multiple new and classic compounds, we found that some compounds, including 1R,4R-3-(6-aminopurin-9-yl)-cyclopentanecarboxylic acid hydroxyamide, potently inhibited type 5 adenylyl cyclase, the major cardiac isoform, but not other isoforms. In normal mouse cardiac myocytes, contraction induced by low ␤-adrenergic receptor stimulation was poorly inhibited with this compound, but the induction of cardiac myocyte apoptosis by high ␤-adrenergic receptor stimulation was effectively prevented by type 5 adenylyl cyclase inhibitors. In contrast, when cardiac myocytes from type 5 adenylyl cyclase knock-out mice were examined, ␤-adrenergic stimulation poorly induced apoptosis. Our data suggest that the inhibition of ␤-adrenergic signaling at the level of the type 5 adenylyl cyclase isoform by P-site inhibitors may serve as an effective method to prevent cardiac myocyte apoptosis induced by excessive ␤-adrenergic stimulation without deleterious effect on cardiac myocyte contraction.Adrenergic stimulation via the sympathetic nervous system is a major mechanism to regulate cardiac function (1). Norepinephrine released from the synaptic terminal binds to ␤-adrenergic receptors (␤-AR), 1 leading to the activation of adenylyl cyclase (AC) and thus the production of cAMP, which initiates a cascade of protein phosphorylation and regulation of many key signaling elements involved in muscle contraction and calcium handling. The immediate outcome of this stimulation is increased cardiac contractility and output. Pharmacological inhibition of this pathway, by the use of ␤-AR antagonists, has been widely utilized to attenuate cardiac function in the clinical setting of treating high blood pressure for several decades. Recently, ␤-AR antagonists have been used in the treatment of heart failure, i.e. in hearts with deteriorated cardiac function. This paradoxical usage of ␤-AR antagonists in heart failure has been rationalized by the protection of the heart from excessive sympathetic stimulation. Under this pathological condition, it is well known that the sympathetic activity is increased markedly (2-4), leading to the remodeling of the heart (5) and, more importantly, the induction of cardiac myocyte apoptosis, as demonstrated by both in vivo and in vitro studies (6, 7). However, a major problem in introducing ␤-AR antagonist therapy is the worsening of cardiac function (8).It is believed that regulating ␤-adrenergic signaling at the level of AC, instead of the receptor, may serve as an alternative to the common ␤-AR regulating therapy, which is most exemplified by direct ...

Antiviral drug vidarabine possessing cardiac type 5 adenylyl cyclase inhibitory property did not affect cardiohemodynamic or electrophysiological variables in the halothane-anesthetized dogs

“…These results suggest that type 5 adenylyl cyclase in the heart may play a minor role in the production of cyclic AMP under physiological condition. However, inhibitory effect of vidarabine on type 5 adenylyl cyclase was confirmed in the presence of isoproterenol, forskolin and/or guanosine 5'-[γ-thio] triphospahate (GTPγS) (Iwatsubo et al, 2004(Iwatsubo et al, , 2012Braeunig et al, 2013). One can speculate that vidarabine might be at least in part expected to improve the pathophysiology of congestive heart failure in the presence of increased adrenergic tone, whereas modification will not be exerted during the absence of such pathological situation, which may become advantage over the conventional Na + channel inhibitors and/or β blockers.…”

“…Vidarabine in a dose of 10-15 mg/kg is clinically recommended for the treatment of herpes simplex encephalitis in the interview form (Sep. 2011, ver.3) from the manufacturer. Moreover, vidarabine has been reported to inhibit type 5 adenylyl cyclase (Iwatsubo et al, 2004), thus to improve the left ventricular ejection fraction and survival rate of experimental mice models of congestive heart failure (Iwatsubo et al, 2012). Recently, vidarabine was shown to suppress atrial fibrillation of mice induced by transesophageal atrial burst pacing during isoproterenol infusion (Suita et al, 2014).…”

Epac increases melanoma cell migration by a heparan sulfate-related mechanism

“…Western blot analysis was performed as we described previously (20). Cells were lysed and sonicated in lysis buffer containing 25 mM Tris ⅐ HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM dithiothreitol, 5% glycerol, phosphatase inhibitor (Sigma), protease inhibitor cocktail (Sigma), and 1 mM NaF.…”

Molecular Details of cAMP Generation in Mammalian Cells: A Tale of Two Systems

“…The same differences between the adenine and sugar binding pockets of sAC and tmACs, which decreases AC's affinity for ATP, should also explain its diminished sensitivity to inhibition by P-site ligands. 117 Interestingly, a new series of P-site inhibitors, such as the compoundPMC-6 (1R,4R-3-(6-aminopurin-9-yl)-cyclopentanecarboxylic acid hydroxyamide), contain a metal chelating moiety 118,119 and can discriminate between some tmAC isoforms; 119 PMC-6 inhibits type II and III modestly, but tmAC V with high potency.…”