Direct stimulation of adenylyl cyclase 9 by the fungicide imidazole miconazole
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In mammals, nine genes encode trans-membrane adenylyl cyclase (tmAC) isoforms that synthesize the intracellular messenger compound cAMP from ATP. As cAMP is produced in virtually all types of cell, isoform-selective modulators of tmAC would have major research and therapeutic potential. This study investigated the effects of fungicide imidazoles previously shown to suppress cAMP production in various tissues on the activities of tmAC isoforms AC1, 2, or 9 stably expressed in human embryonic kidney 293 cells. Intact cells, as well as crude membranes, were exposed to various imidazoles or known stimulators of tmAC and the ensuing changes in the production of cAMP analyzed. In crude membranes, the activity of AC9 in the presence of GDP-β-S was enhanced by miconazole with an EC50 of ~ 8 μM, while AC1 and AC2 were inhibited with an IC50 of ~ 20 μM. Clotrimazole (10–100 μM) was an inhibitor of all the ACs tested. Substrate saturation analysis indicated that miconazole increased the Vmax of AC9 by 3-fold while having no effect on the Km. In intact cells, the effect of miconazole on cAMP production through AC9 was additive with that of isoproterenol. The stimulation of cAMP production by miconazole was inhibited by Ca2+, and this could be prevented by the calcineurin blocker FK506. In sum, activation of AC9 by miconazole is through a mechanism distinct from that of forskolin, activated G proteins, or the COOH-terminal mediated autoinhibition. However, it is subject to the AC9 isoform-specific inhibition by Ca2+/calcineurin. Differential modulation of mammalian tmAC paralogs appears to be achievable by an imidazole with phenylated side chains. Optimization of the lead compound and exploration of the underlying mechanism(s) of action in more detail could exploit this further.
KeywordsAdenosine 3′5′monophosphate Trans-membrane adenylyl cyclase Adenylyl cyclase Ca2+/calcineurin
We thank Dr. Janice Paterson and Mrs. Susan M. Smith for HEK293 cells expressing human AC9, Dr. A. Friedrich, Fujisawa GmbH, Munich, Germany for FK506, Dr. William Parsons, Merck and Co, Rahway N.J., for L685,818, and Dr. H. Wachtel, Schering AG., Berlin, Germany for rolipram.
AP and JS carried out experiments, collated and analyzed data, FAA designed and carried out experiments, analyzed and collated data, wrote the manuscript. All authors have agreed on the text of the submitted manuscript.
This work was supported by the Medical Research Council, UK and the Servier-Egis research co-operation agreement.
Compliance with ethical standards
Conflict of interest
AP and FAA were employees of Egis Pharmaceuticals PLC, Budapest, Hungary.
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