Applicability of green fluorescence protein in the study of endothelin converting enzyme-1c trafficking.

Endothelin-1 (ET-1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor big endothelin-1 by endothelin converting enzyme-1 (ECE-1). Production of ET-1 is thought to be dependent upon the expression of ECE-1 at the cell surface. Therefore, mechanisms inducing the trafficking of ECE-1 to the cell surface have been the focus of recent research. This research has identified phosphorylation of the cytoplasmic region of ECE-1 as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE-1 to monitor phosphorylation induced trafficking of ECE-1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE-1, and hence the suitability of GFP or any other protein tag in studying ECE-1 distribution and trafficking. ECE-1c is the most widely expressed isoform in endothelial cells. We therefore expressed ECE-1c with a GFP tag either at the N or C-terminus of ECE-1c. Catalytic activity and effect on protein kinase C (PKC) induced phosphorylation was compared between the two chimeras and wild-type ECE-1c. Our results indicate that positioning of the GFP tag on the C-terminus abrogates activity without effecting PKC-induced phosphorylation. However, GFP tag on the N-terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE-1c distribution and trafficking.

Protein Kinase C recognition sites in the cytoplasmic domain of Endothelin Converting Enzyme-1c.

Endothelin Converting Enzyme-1 (ECE-1) is essential for the production of the potent vasoconstrictor Endothelin-1 (ET-1). The activation of Protein Kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) can increase ECE-1 phosphorylation, which in turn promotes ECE-1c trafficking to the cell surface where ET-1 production occurs. This study has identified the specific residues in the N-terminal cytoplasmic tail of ECE-1c isoform that are phosphorylated upon the activation of PKC. Levels of phosphorylation are expressed as a % phosphorylation in untreated CHO-K1 cells. We transfected CHO-K1 cells with wild type and mutant forms of ECE-1c (Ala(4)-ECE-1c, Ala(35)ECE-1c and Ala(4/35)ECE-1c) to confirm the involvement of Thr(4) and Ser(35) residues in PMA induced phosphorylation of ECE-1c. Phosphorylation of wild type ECE-1c increased in response to PMA treatment (150±13%, unpaired t-test, P<0.05, significantly different compared to untreated control). The two single mutants and one combined mutant significantly reduced the PMA induced phosphorylation (103-117±6-13%; unpaired t-test; n=8; P<0.05 significantly different compared to untreated control). The mutations had no effect on the basal ECE-1c phosphorylation. In addition, they had no effect on the catalytic activity as evidenced by the similar rate of substrate cleavage compared to wild type. This study is the first to confirm the residues phosphorylated upon the activation of PKC by PMA. The results complete a gap in our understanding of the mechanism(s) behind PKC induced trafficking of ECE-1.

Assessment of the pharmacological properties of 5-methoxyindole derivatives at 5-HT4 receptors.

OBJECTIVES: The aim was to examine the biological activity of 5-methoxytryptamine derivatives at the 5-hydroxytryptamine (5-HT)(4) receptor to explore the effect of substitution on the aliphatic amine of the 5-methoxyamine scaffold. METHODS: Three compounds were tested for affinity at the 5-HT(4) receptor by radioligand binding and functional activity using guinea-pig ileum and human colon circular muscle preparations and also in the mouse whole gut transit test. KEY FINDINGS: The three compounds all had agonist properties at the 5-HT(4) receptor but their efficacy differed in the different functional tests. Compound 3 had the highest affinity for the 5-HT(4) receptor and was a full agonist at relaxing human colon circular muscle with efficacy closest to 5-HT. Compounds 1 and 2 were partial agonists in this assay with lower efficacies; compound 2 was a full agonist in the guinea-pig ileum assay whereas compound 3 was a partial agonist. Compounds 1 and 2 also showed activity in the mouse gut transit assay while compound 3 had no activity. CONCLUSIONS: Of the compounds tested, compound 3 was the most promising 5-HT(4) receptor agonist and the results highlight the value of using human tissue in functional tests when assessing compounds for potential activity.

Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically.

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function.

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon.

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.

Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1.

The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone.

Quenched fluorescent substrate-based peptidase assays.

The use of specific quenched fluorescent substrates (QFS) provides a rapid and sensitive method to measure peptidase activity, and is readily adaptable to high-throughput screening of potential peptidase inhibitors. In this chapter, we discuss general considerations for the development of QFS assays, and describe in detail an assay protocol for the mammalian metallopeptidase, endothelin-converting enzyme.