betadex and mastoparan

Lipid rafts and caveolae are microdomains in the cell membranes, which contain cholesterol, glycolipids, and sphingomyelin. While caveolae are relatively stable because caveolin, an integral protein, supports the structure, lipid rafts are considered to be unstable, being dynamically produced and degraded. Recent studies have reported that lipid rafts contain many signaling molecules, such as glycosylphosphatidylinositol-anchored proteins, acylated proteins, G-protein-coupled receptors (GPCRs), trimeric and small G-proteins and their effectors, suggesting that the lipid rafts have an important role in receptor-mediated signal transduction. Therefore drugs that modify the composition of lipid rafts might influence the efficacy of cellular signal transduction. In this review, we demonstrate the role of lipid rafts in GPCR-G-protein signaling and also present our recent results showing that the wasp toxin mastoparan modifies G(q/11)-mediated phospholipase C activation through the interaction with gangliosides in lipid rafts.

Topics: beta-Cyclodextrins; Cholesterol; Gangliosides; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Membrane Microdomains; Peptides; Receptors, G-Protein-Coupled; Signal Transduction; Type C Phospholipases; Wasp Venoms

Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon.. Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I(sc)) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt.. Luminal addition of methyl-β-cyclodextrin (10 mm) and mastoparan (30 μm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I(sc) , but did not affect increases in I(sc) induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 μm), a Gα(q/11) -selective inhibitor, markedly inhibited propionate-induced increase in I(sc) , but did not affect I(sc) responses to ACh and EFS. Both methyl-β-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and Gα(q/11) , but not GPR43. Uncoupling of Gα(q/11) from flotillin-1 in lipid rafts occurred under desensitization of the I(sc) response to propionate.. These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via Gα(q/11) .

Topics: Acetylcholine; Animals; beta-Cyclodextrins; Blotting, Western; Centrifugation, Density Gradient; Cholesterol; Colon; Electric Stimulation; GTP-Binding Protein alpha Subunits, Gq-G11; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Intestinal Secretions; Male; Membrane Microdomains; Membrane Potentials; Membrane Proteins; Peptides; Peptides, Cyclic; Propionates; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Wasp Venoms