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Earlier we reported defects in D1 receptor function in renal proximal tubules (RPTs) of aged Fischer 344 (F344) and obese Zucker rats. However, the defects in the receptor function in RPTs of obese Zucker rats do not pass onto primary cultures of RPTs from these animals. Here, we determined whether the defects in D1 receptor function in RPTs of aged F344 rats pass onto the primary cultures. RPTs from aged (24-mo) and adult (6-mo) F344 rats were grown into primary cultures. The microscopic studies showed that ceils in cultures from adult and old rats were healthy as determined by the shape and size of the cells and nuclei. D1 receptor agonist SKF-38393 produced inhibition of [sup.86]Rb (rubidium) uptake, index of Na-K-ATPase activity, in ceils from adult rats, but this was reduced in old rats. Also, SKF-38393 increased the [[sup.35]S]GTP3[gamma]S binding, index of receptor activation, in the membranes of cells from adult rats but to a lesser extent from old rats. Furthermore, there was a downward trend in the levels of D 1 receptor numbers and in the receptor proteins in old rats. Interestingly, [gp.sup.91phox] subunit of NADPH oxidase and cellular protein carbonyl levels (oxidative stress marker) were higher in cultures from old rats. These results show that RPTs from adult and old F344 rats grow into epithelial cells in cultures. Furthermore, cells in cultures from old rats are at a higher level of oxidative stress, which may be contributing to the reduced D1 receptor function in the cells from old compared with adult rats. dopamine; NADPH oxidase; G protein-coupled receptor; aging

Abstract Background Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E1 (PGE1) and E2 (PGE2) can be identified in the water-soluble non-purified fraction. PGE1 is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE2 is an inflammatory molecule. Methods We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP1 receptor stably expressed in HEK293 cells (human embryonic kidney). PGE1 and PGE2 were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened. Results After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration) using fluorescence microscopy. Conclusions Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, PGE1 and PGE2.

Background: Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E1 (PGE1) and E2 (PGE2) can be identified in the water-soluble non-purified fraction. PGE1 is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE2 is an inflammatory molecule. Methods: We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP1 receptor stably expressed in HEK293 cells (human embryonic kidney). PGE1 and PGE2 were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened. Results: After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration) using fluorescence microscopy. Conclusions: Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, PGE1 and PGE2. [ABSTRACT FROM AUTHOR]

Abstract: A peptide constrained to a conformation of second extracellular loop of human prostaglandin-E2 (PGE2) receptor subtype3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE2 were first identified by NMR spectroscopy. The results were used as a guide for site-directed mutagenesis of the hEP3 protein. The S211L and R214L mutants expressed in HEK293 cells lost binding to [3H]PGE2. This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE2 recognition, and implied that the corresponding residues in other subtype receptors could be important to distinguish the different configurations of PGE2 ligand recognition sites. [Copyright &y& Elsevier]