Characterization of Mouse Heart Adenylyl Cyclase

Abstract

Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces ␤-adrenoceptor-stimulated Ca 2ϩ inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg 2ϩ were similar. Moreover, the inhibitory profile of eight 2Ј(3Ј)-O-(N-methylanthraniloyl) (MANT)-nucleoside 5Ј-([␥-thio])triphosphates on mouse heart in the presence of Mg 2ϩ was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K i values in the 15 to 25 nM range in the presence of Mg 2ϩ and in the 1 to 5 nM range in the presence of Mn 2ϩ . However, in the presence of Mn 2ϩ , we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg 2ϩ , AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn 2ϩ masking AC5 effects were problems.

Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors ofBordetella pertussisAdenylyl Cyclase Toxin

“…In accordance with previous studies (Gille et al, 2004;Göttle et al, 2009), AC2 was the AC isoform with the lowest inhibitor sensitivity because of the exchange of A409P and V1108I in AC1 and AC5 versus AC2 (Mou et al, 2005). (M)ANT-NTPs with the purine base hypoxanthine had the highest potency.…”

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…However, identifying AC isoform distribution remains a challenge (Antoni et al, 2006). First and foremost, there is a dearth of well-characterized isoform-specific antibodies (Iwamoto et al, 2003;Antoni et al, 2006;Sadana and Dessauer, 2009;Göttle et al, 2009). In some cases, antibodies have been used along with other molecular (mRNA measures) or biochemical (functional assays) approaches revealing partially consistent results Johnston et al, 2004;Liu et al, 2004;Göttle et al, 2009).…”

“…Although organ membranes contain numerous AC isoforms, this system is relatively close to the physiologic situation. By combining physiologic and pharmacological activators, inhibitors, RT-PCR analysis of expressed mRNA and, if available, antibodies, analysis of ACs in organ membranes can be quite informative and yield valuable information on functionally dominant AC isoforms (Göttle et al, 2009;Erdorf and Seifert, 2011). Experiments with native organs have been successfully complemented with gene knockout studies and transgene overexpression studies in mice Sadana and Dessauer, 2009).…”

Human Bronchial Smooth Muscle Cells Express Adenylyl Cyclase Isoforms 2, 4, and 6 in Distinct Membrane Microdomains

“…However, other pharmacological approaches for defining AC isoform expression are available and are particularly applicable for characterizing AC isoforms in tissues, whereas using these antibodies is more problematic. AC isoforms can be defined by rank order of sensitivity to various nucleotide and non-nucleotide inhibitors or by activation by isoform-selective derivatives of forskolin (Gille et al, 2004;Pinto et al, 2008;Göttle et al, 2009). Whereas no single approach can be used to reliably characterize the AC isoforms expressed in a given system, a combination of molecular and pharmacological tools can be deployed to achieve this goal.…”