Pharmacological effects of ephedrine alkaloids on human alpha(1)- and alpha(2)-adrenergic receptor subtypes.

Ephedra species of plants have both beneficial and adverse effects primarily associated with the presence of ephedrine alkaloids. Few reports have appeared that examine the direct actions of ephedrine alkaloids on human subtypes of adrenergic receptors (ARs). In the present study, ephedrine alkaloids were evaluated for their binding affinities on human alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, and alpha(2C)-AR subtypes expressed in HEK and Chinese hamster ovary cells. Cell-based reporter gene assays were used to establish functional activity of ephedrine alkaloids at alpha(1A)-, alpha(2A)-, and alpha(2C)-ARs. The data showed that ephedrine alkaloids did not activate alpha(1)- and alpha(2)-ARs and that they antagonized the agonist-mediated effects of phenylephrine and medetomidine on alpha(1)- and alpha(2)-ARs, respectively. As in the binding studies, 1R,2R- and 1R,2S-ephedrine showed greater functional antagonist activity than the 1S,2R- and 1S,2S-isomers. The rank order of affinity for the isomers was 1R,2R > 1R,2S > 1S,2R > 1S,2S. The rank order of potencies of alkaloids containing a 1R,2S-configuration was norephedrine > or = ephedrine >> N-methylephedrine. These studies have demonstrated that orientation of the beta-hydroxyl group on the ethylamino side chain and the state of N-methyl substitution are important for alpha-AR binding and functional activity of the ephedrine alkaloids. In conclusion, the ephedrine isomers and analogs studied did not exhibit any direct agonist activity and were found to possess moderate antagonist activities on cloned human alpha-ARs. The blockade of presynaptic alpha(2A)- and alpha(2C)-ARs may have a pharmacological role in the direct actions of Ephedra alkaloids.

Pterostilbene, a new agonist for the peroxisome proliferator-activated receptor alpha-isoform, lowers plasma lipoproteins and cholesterol in hypercholesterolemic hamsters.

Resveratrol, a stilbenoid antioxidant found in grapes, wine, peanuts and other berries, has been reported to have hypolipidemic properties. We investigated whether resveratrol and its three analogues (pterostilbene, piceatannol, and resveratrol trimethyl ether) would activate the peroxisome proliferator-activated receptor alpha (PPARalpha) isoform. This nuclear receptor is proposed to mediate the activity of lipid-lowering drugs such as the fibrates. The four stilbenes were evaluated at 1, 10, 100, and 300 microM along with ciprofibrate (positive control), for the activation of endogenous PPARalpha in H4IIEC3 cells. Cells were transfected with a peroxisome proliferator response element-AB (rat fatty acyl CoA beta-oxidase response element)-luciferase gene reporter construct. Pterostilbene demonstrated the highest induction of PPARalpha showing 8- and 14-fold increases in luciferase activity at 100 and 300 microM, respectively, relative to the control. The maximal luciferase activity responses to pterostilbene were higher than those obtained with the hypolipidemic drug, ciprofibrate (33910 and 19460 relative luciferase units, respectively), at 100 microM. Hypercholesterolemic hamsters fed with pterostilbene at 25 ppm of the diet showed 29% lower plasma low density lipoprotein (LDL) cholesterol, 7% higher plasma high density lipoprotein (HDL) cholesterol, and 14% lower plasma glucose as compared to the control group. The LDL/HDL ratio was also statistically significantly lower for pterostilbene, as compared to results for the control animals, at this diet concentration. Results from in vitro studies showed that pterostilbene acts as a PPARalpha agonist and may be a more effective PPARalpha agonist and hypolipidemic agent than resveratrol. In vivo studies demonstrate that pterostilbene possesses lipid and glucose lowering effects.

Evaluation of beta-adrenergic receptor subtypes in the human prostate cancer cell line-LNCaP.

The present study was undertaken to determine the effects of catecholamines, agonists, and antagonists of beta-adrenergic receptors (AR) in the LNCaP cell line. Changes in cellular cyclic adenosine-3',5'-monophosphate (cAMP) levels were quantified by the use of a 6 cAMP response element (CRE)-luciferase reporter gene assay. LNCaP cells were transiently transfected with this gene construct, incubated in 96-well microtiter plates for 24 hr, and then treated with beta-AR agonists and/or antagonists for 4 hr. The rank order of potency for catecholamines and known beta-AR agonists was terbutaline(3.31 nM)>isoproterenol(8.31 nM)> or =fenoterol(15 nM)=epinephrine(16.2 nM)>norepinephrine(77.5 nM)>BRL-37344 [(R(*),R(*))-(+/-)4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy acetic acid, sodium salt] (1000 nM)>dobutamine(1770 nM)>CGP12177 (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazole-2-one hydrochloride) (inactive). The non-selective beta(1)-/-beta(2)-AR antagonists; propranolol and CGP 12177, at 10(-7)M, inhibited luciferase activity induced by these agonists by 80-96%. Propranolol blocked isoproterenol-induced luciferase responses in a competitive manner (K(B)=1.4 nM). In addition, isoproterenol-activated luciferase expression was blocked more potently by ICI 118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethy) amino]-2-butanol], a beta(2)-AR antagonist than by ICI 89,406 [(+/-)-N-[2-[3-(2-cyanophenoxy-)]-2-hydroxypropylamino]ethyl-N-phenylurea], a beta(1)-AR antagonist, giving K(B) values of 1.07 and 161nM, respectively. These results suggest that the beta(2)-AR is the major subtype mediating catecholamine-induced cAMP changes in LNCaP cells.