Camp Pathway Modifiers Alter Lipid Bilayer Properties

Abstract

Cyclic adenosine monophosphate (cAMP) is a major intracellular messenger. Within the cAMP pathways, several players mediate cAMP production and degradation. The two main producers of cAMP are the transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), genetically distinct enzymes producing the same end product. The relative contributions of sAC and tmAC to cAMP production usually are explored using pharmacological interventions. Many of the compounds used in such studies are hydrophobic, and (for a given modifier) the range of concentrations that are used to obtain the same effect vary widely and inconsistently. These compounds are also known to alter membrane protein function at concentrations that overlap with those used to target cAMP metabolism. We therefore characterized the lipid bilayer-perturbing effects of modifiers of sAC and tmAC and the phosphodiesterases that catabolize cAMP. We used gramicidin-based stopped flow spectrofluorometry to measure the bilayer-modifying (off-target) effects of compounds used to manipulate cAMP metabolism. Of 16 compounds tested thus far, at least four perturb the bilayer at commonly used concentrations. Among the bilayer-active compounds are KH7 a sAC inhibitor, forskolin a pan-cyclase activator, 1,9-dideoxy forskolin a cyclase-inactive forskolin derivative, and isobutylmenthylxanthine, a non-selective phosphodiesterase inhibitor. These compounds thus may alter cell function by being promiscuous modifiers of membrane proteins⎯in addition to their effects on cAMP metabolism. There is no correlation between the bilayer perturbing effect and the calculated partition coefficients, indicative of an unexpected complexity that should be taken into consideration when planning experiments with hydrophobic compounds. Our overarching conclusion is that small hydrophobic molecules should be used with care, and that any effects that are observed only at concentrations that are two-to-three times higher than the concentrations for half-maximal effect (in terms of modifying the intended target in vitro) should be interpreted with caution.