Small Ligands Modulating the Activity of Mammalian Adenylyl Cyclases: A Novel Mode of Inhibition by Calmidazolium

Abstract

Molecular cloning of membrane-spanning mammalian adenylyl cyclases (ACs) has led to the discovery of nine different isotypes, making ACs potentially useful therapeutic targets. This study investigated the mechanism by which fungicidal nitroimidazole compounds modulate AC activity. Current evidence indicates that biological control of AC activity occurs through the cytosolic domains. Hence, full-length ACII, ACIX, and recombinant fusion proteins composed of the cytoplasmic loops of human ACIX or the first and second cytoplasmic loops of rat ACV and ACII, respectively, were expressed in human embryonic kidney 293 cells. The AC activities of the respective proteins were characterized, and their modulation by nitroimidazoles was investigated. Calmidazolium inhibited the activities of both full-length ACs and soluble fusion proteins (IC 50 , ϳ10 M). Inhibition of ACIX by calmidazolium was mediated by direct interaction with the catalytic core in a noncompetitive fashion. ACIX was essentially insensitive to 2Ј-deoxyadenosine 3Ј-monophosphate, a known blocker of AC activity. The ACV-ACII fusion protein was inhibited by calmidazolium (IC 50 , ϳ20 M) as well as by 2Ј-deoxyadenosine 3Ј-AMP (IC 50 , ϳ2 M), in a manner indicating independent mechanisms of action. Taken together, the data demonstrate that ACIX is insensitive to adenosine analogs and that calmidazolium inhibits AC activity by a novel, noncompetitive mechanism.

Biochemical properties of heterologously expressed and native adenylyl cyclases from the honeybee brain (Apis mellifera L.)

“…Immunostaining of honeybee brain with specific AmAC3 antibodies revealed a prominent expression of AmAC3 in the antennal lobes. When enzymatic activity was inhibited with P-site inhibitors (adenine nucleoside-3 0 -polyphosphates; Dessauer and Gilman, 1997;Haunso et al, 2003) the same rank order of potency was observed for heterologously expressed AmAC3 and ACs present in antennal lobes (2 0 , 5 0 dd3 0 ATP > 3 0 AMP > 2 0 deoxyadenosine). The activity of AmAC3 was also impaired by Ca 2þ /CaM, which was independent of CaM kinase II (CaMKII) activation.…”

Calcium-independent phospholipase A2 mediates store-operated calcium entry in rat cerebellar granule cells

“…Preincubation of BEL almost completely blocked the CMZ-induced Ca 2+ rise to 9¡1% DF (pv0.005, n5218/4), which was even significantly smaller than in control cells (pv0.005), in which the re-addition of Ca 2+ induced a Ca 2+ rise of 56¡3% DF (pv0.005, n5154/5, Figure 4A and B). Since CMZ has been reported to inhibit adenylyl cyclase (AC) activity, though at higher concentrations than used in the present study (35), we tested whether the CMZ-induced Ca 2+ influx upon readdition of Ca 2+ can be mimicked by inhibition of AC activity. We used the AC inhibitor SQ 22536 (200 mM) in the absence of CMZ for 15 min before re-adding Ca 2+ .…”

Regulation of Store-Operated Calcium Entry by Calcium-Independent Phospholipase A2in Rat Cerebellar Astrocytes

“…CMZ has been shown to inhibit the activity of adenylyl cyclase (AC), albeit at greater concentrations than those used in the present study (Haunso et al, 2003). We tested whether the CMZ-induced augmentation of the Ca 2ϩ transient during Ca 2ϩ readdition can be mimicked by the inhibition of AC activity.…”