Exploring the mechanism of PPARγ phosphorylation mediated by CDK5.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with a key role in metabolic processes and is target of CDK5 kinase phosphorylation at S245 (S273 in PPARγ isoform 2), thereby inducing insulin resistance. A remarkable effort has been addressed to find PPARγ ligands that inhibit S245 phosphorylation, but the poor understanding in this field challenges the design of such ligands. Here, through computational and biophysical methods, we explored an experimentally validated model of PPARγ-CDK5 complex, and we presented K261, K263 or K265, which are conserved in mammals, as important anchor residues for this interaction. In addition, we observed, from structural data analysis, that PPARγ ligands that inhibit S245 phosphorylation are not in direct contact with these residues; but induce structural modifications in PPARγ:CDK5/p25 interface. In summary, our PPARγ and CDK5/p25 interaction analyses open new possibilities for the rational design of novel inhibitors that impair S245 phosphorylation.

ß-Citronellol, an alcoholic monoterpene with inhibitory properties on the contractility of rat trachea.

ß-Citronellol is an alcoholic monoterpene found in essential oils such Cymbopogon citratus (a plant with antihypertensive properties). ß-Citronellol can act against pathogenic microorganisms that affect airways and, in virtue of the popular use of ß-citronellol-enriched essential oils in aromatherapy, we assessed its pharmacologic effects on the contractility of rat trachea. Contractions of isolated tracheal rings were recorded isometrically through a force transducer connected to a data-acquisition device. ß-Citronellol relaxed sustained contractions induced by acetylcholine or high extracellular potassium, but half-maximal inhibitory concentrations (IC50) for K(+)-elicited stimuli were smaller than those for cholinergic contractions. It also inhibited contractions induced by electrical field stimulation or sodium orthovanadate with pharmacologic potency equivalent to that seen against acetylcholine-induced contractions. When contractions were evoked by selective recruitment of Ca2+ from the extracellular medium, ß-citronellol preferentially inhibited contractions that involved voltage-operated (but not receptor-operated) pathways. ß-Citronellol (but not verapamil) inhibited contractions induced by restoration of external Ca2+ levels after depleting internal Ca2+ stores with the concomitant presence of thapsigargin and recurrent challenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacin or tetraethylammonium did not change the relaxing effects of ß-citronellol. Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) receptors with selective antagonists caused no change in the effects of ß-citronellol. In conclusion, ß-citronellol exerted inhibitory effects on rat tracheal rings, with predominant effects on contractions that recruit Ca2+ inflow towards the cytosol by voltage-gated pathways, whereas it appears less active against contractions elicited by receptor-operated Ca2+ channels.

ß-Citronellol, an alcoholic monoterpene with inhibitory properties on the contractility of rat trachea

β-Citronellol is an alcoholic monoterpene found in essential oils such Cymbopogon citratus (a plant with antihypertensive properties). β-Citronellol can act against pathogenic microorganisms that affect airways and, in virtue of the popular use of β-citronellol-enriched essential oils in aromatherapy, we assessed its pharmacologic effects on the contractility of rat trachea. Contractions of isolated tracheal rings were recorded isometrically through a force transducer connected to a data-acquisition device. β-Citronellol relaxed sustained contractions induced by acetylcholine or high extracellular potassium, but half-maximal inhibitory concentrations (IC50) for K+-elicited stimuli were smaller than those for cholinergic contractions. It also inhibited contractions induced by electrical field stimulation or sodium orthovanadate with pharmacologic potency equivalent to that seen against acetylcholine-induced contractions. When contractions were evoked by selective recruitment of Ca2+ from the extracellular medium, β-citronellol preferentially inhibited contractions that involved voltage-operated (but not receptor-operated) pathways. β-Citronellol (but not verapamil) inhibited contractions induced by restoration of external Ca2+ levels after depleting internal Ca2+ stores with the concomitant presence of thapsigargin and recurrent challenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacin or tetraethylammonium did not change the relaxing effects of β-citronellol. Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) receptors with selective antagonists caused no change in the effects of β-citronellol. In conclusion, β-citronellol exerted inhibitory effects on rat tracheal rings, with predominant effects on contractions that recruit Ca2+ inflow towards the cytosol by voltage-gated pathways, whereas it appears less active against contractions elicited by receptor-operated Ca2+ channels.

(-)-α-Bisabolol inhibits preferentially electromechanical coupling on rat isolated arteries.

Previous findings enable us to hypothesize that (-)-α-bisabolol acts as inhibitor of voltage-dependent Ca(2+) channels in smooth muscle. The current study was aimed at consolidating such hypothesis through the recording of isometric tension, measurement of intracellular Ca(2+) as well as discovery of channel target using in silico analysis. In rat aortic rings, (-)-α-bisabolol (1-1000 µM) relaxed KCl- and phenylephrine-elicited contractions, but the IC50 differed significantly (22.8 [17.6-27.7] and 200.7 [120.4-334.6] µM, respectively). The relaxation of phenylephrine contractions remained unaffected by l-NAME, indomethacin, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, tetraethylammonium, glibenclamide or KT-5720. Under Ca(2+)-free conditions, (-)-α-bisabolol did not alter the contractions evoked by phenylephrine or caffeine whereas it reduced those evoked by CaCl2 in KCl-, but not in PHE-stimulated preparations. Furthermore, it did not significantly alter the contractions evoked by phorbol 12,13-dibutyrate or induced by the extracellular Ca(2+) restoration in cyclopiazonic acid-treated preparations. In mesenteric rings loaded with Fluo-4 AM, (-)-α-bisabolol blunted the tension and the cytosolic levels of Ca(2+) in response to K(+) but not to norepinephrine. Silico docking analysis of the Cavß2a subunit of voltage-dependent Ca(2+) channel indicated putative docking sites for (-)-α-bisabolol. These findings reinforce the ability of (-)-α-bisabolol to inhibit preferentially contractile responses evoked by Ca(2+) influx through voltage-dependent Ca(2+) channels.

Talking about bioelectrical potentials using rings of the mesenteric artery without glass micropipettes.

In the present study, a practical activity is proposed to adopt an experimental approach to demonstrate the relationship between the equilibrium potential for K(+) and transmembrane electrical potential without glass micropipettes. A conventional setup for recording contractile activity of isolated smooth muscle preparations was used based on the events elegantly described by Somlyo and Somlyo in the 1960s. They showed that, in response to a given stimulus, smooth muscle cells may contract, recruiting electromechanical or pharmacomechanical coupling by mechanisms that involve, or not, changes in transmembrane potential, respectively. By means of contractions and relaxations of a ring-like preparation from the rat mesenteric artery, it is possible to observe the functional consequences of handling K(+) concentration in the extracellular compartment and the effects caused by opening K(+) channels in that preparation, which are significant when the cell membrane establishes an electrical potential difference between intra- and extracellular compartments (driven mainly by K(+) permeability under resting conditions). The effects observed by students fit well with values predicted by Nernst and Goldman-Hodgin-Katz equations, and we demonstrated that the activity is able to improve students' comprehension regarding basic principles of bioelectricity.