The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid CB₁ receptor in vitro

This investigation focused primarily on the interaction of two benzophenanthridine alkaloids (chelerythrine and sanguinarine), piperonyl butoxide and (S)-methoprene with G-protein-coupled cannabinoid CB(1) receptors of mouse brain in vitro. Chelerythrine and sanguinarine inhibited the binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at low micromolar concentrations (IC(50)s: chelerythrine 2.20 μM; sanguinarine 1.10 μM). The structurally related isoquinoline alkaloids (berberine and papaverine) and the phthalide isoquinoline ((-)-β-hydrastine) were either inactive or considerably below IC(50) at 30 μM. Chelerythrine and sanguinarine antagonized CP-55940-stimulated binding of [(35)S] GTPγS to the G-protein (IC(50)s: chelerythrine 2.09 μM; sanguinarine 1.22 μM). In contrast to AM251, both compounds strongly inhibited basal binding of [(35)S]GTPγS (IC(50)s: chelerythrine 10.06 μM; sanguinarine 5.19μM). Piperonyl butoxide and S-methoprene inhibited the binding of [(3)H]CP-55940 (IC(50)s: piperonyl butoxide 8.2 μM; methoprene 16.4 μM), and also inhibited agonist-stimulated (but not basal) binding of [(35)S]GTPγS to brain membranes (IC(50)s: piperonyl butoxide 22.5 μM; (S)-methoprene 19.31 μM). PMSF did not modify the inhibitory effect of (S)-methoprene on [(3)H]CP-55940 binding. Our data suggest that chelerythrine and sanguinarine are efficacious antagonists of G-protein-coupled CB(1) receptors. They exhibit lower potencies compared to many conventional CB(1) receptor blockers but act differently to AM251. Reverse modulation of CB(1) receptor agonist binding resulting from benzophenanthridines engaging with the G-protein component may explain this difference. Piperonyl butoxide and (S)-methoprene are efficacious, low potency, neutral antagonists of CB(1) receptors. Certain of the study compounds may represent useful starting structures for development of novel/more potent G-protein-coupled CB(1) receptor blocking drugs.     

Selection of insensitive acetylcholinesterase as a resistance mechanism in Aedes aegypti (Diptera: Culicidae) from Santiago de Cuba

A sample of Aedes aegypti (L.) (Diptera: Culicidae) from Santiago de Cuba, Cuba, with a high level of propoxur resistance compared with the reference susceptible Rockefeller strain (12.60 x at the 50% lethal concentration [LC50] and 18.08 at the 90% lethal concentration [LC90]), with a 4.3% frequency of insensitive acetylcholinesterase (AChE) frequency, was subjected to propoxur selection for 13 successive generations to increase the frequency of this resistance mechanism in Ae. aegypti. High resistance to propoxur was developed during this selection (41.73-fold), and the frequency of insensitive AChE mechanism was increased 13.25-fold. Other mechanisms (overproduced esterases, glutathione transferases, or monooxygenases) were not detected in the propoxur-selected strain. The selection of an insensitive AChE resistance mechanism in Ae. aegypti has important implications and will be a valuable resource for genetic studies and molecular characterization of the ace gene mutation(s) associated with insecticide resistance in Ae. aegypti.     

Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists

Background and purpose:

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3′-5′ cyclic guanosine monophosphate (cGMP). Ca²⁺-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC₅₀ values of 11, 80 and 180 µM respectively. Here we sought to elucidate the underlying mechanism(s).

Experimental approach:

We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.

Key results:

Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca²⁺ concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC₅₀= 10 µM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V_max and K_m, indicating that it acts in an uncompetitive manner.

Conclusions and implications:

The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.     

The Use of Dietary Supplements and Amino Acid Restriction Interventions to Reduce Frailty in Pre-Clinical Models

Frailty is a state of accelerated aging that increases susceptibility to adverse health outcomes. Due to its high societal and personal costs, there is growing interest in discovering beneficial interventions to attenuate frailty. Many of these interventions involve the use of lifestyle modifications such as dietary supplements. Testing these interventions in pre-clinical models can facilitate our understanding of their impact on underlying mechanisms of frailty. We conducted a narrative review of studies that investigated the impact of dietary modifications on measures of frailty or overall health in rodent models. These interventions include vitamin supplements, dietary supplements, or amino acid restriction diets. We found that vitamins, amino acid restriction diets, and dietary supplements can have beneficial effects on frailty and other measures of overall health in rodent models. Mechanistic studies show that these effects are mediated by modifying one or more mechanisms underlying frailty, in particular effects on chronic inflammation. However, many interventions do not measure frailty directly and most do not investigate effects in both sexes, which limits their applicability. Examining dietary interventions in animal models allows for detailed investigation of underlying mechanisms involved in their beneficial effects. This may lead to more successful, translatable interventions to attenuate frailty.