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Background Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methods β 2 AR-transfected cells and cells containing endogenous β 2 AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β 2 AR phosphorylation and β 2 AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β 2 AR phosphorylation and internalization. Results Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β 2 ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β 2 AR internalization. Conclusion Amphipathic tastants significantly amplify β 2 AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General Significance Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2’s kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well. [ABSTRACT FROM AUTHOR]

Abstract: To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as l-cysteine, and thereby internalization of the liposomes was significantly facilitated. When l-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells. [Copyright &y& Elsevier]

Abstract: Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM2-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM2-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk−). After 72h incubation at 37°C, TdR showed significant antiproliferative activity (IC50 =27μM) against CEM cells but was weakly effective (IC50 =730μM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5′-monophosphate (POM2-dTMP) was equally inhibitory (IC50 =5μM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-3H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk− with 5μM POM2-[methyl-3H]dTMP. The incorporation of [methyl-3H]TdR into HClO4-insoluble cell residue by CEM tk− was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk− incorporated radioactivity from POM2-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM2-dTMP can penetrate into cells and serve as a source of dTMP. [Copyright &y& Elsevier]