G protein-coupled receptors mediate coronary flow- and agonist-induced responses via lectin-oligosaccharide interactions

Abstract

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, β1-adrenergic, β3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction.

Flow enhances the agonist-induced responses of the bradykinin receptor (B2) because of its lectinic nature: Role of its oligosaccharide environment

“…Synthesis of the soluble oligosaccharide polymer: Well established procedures in our laboratory were utilized [8,12,14,16,26,27]. Briefly, glucose polymer, 70 kDa dextran, was dissolved in NaCO 3 (0.5M, pH 11), mixed with sufficient divinyl sulfone (DVS), so each of the three -OH groups per glucose moiety reacted with one of the two vinyl moieties of each DVS, leaving one free vinyl moiety per each DVS molecule.…”

“…As negative control, no sugars were added for binding but replaced by large amounts of ethanolamine. The negative control polymers do not have a physiological effect and do not bind to the endothelial surface layer [8,12,16,26,28].…”

“…Flow sensing properties likely reside in the luminal endothelial surface; constituted by the cell membrane and glycocalyx which is acted by flow [6][7][8][9][10][11][12][13]. We have shown that flowinduced responses require endothelial surface layer oligosaccharides and oligosaccharide-recognizing proteins, lectins because a) specific enzymatic hydrolysis of endogenous oligosaccharides or their binding to exogenous lectins exactly alter flow-induced responses and b) infusion of diverse exogenous oligosaccharides that bind to endothelial surface lectins alter the effects of flow [8,11,12].…”

“…Our extensive work in the heart has established that coronary flow regulates diverse cardiac and vascular functions, and these flow sensing effects require the presence of various endothelial surface specific oligosaccharides and lectins among which there are several G-protein coupled receptors [12,14]. Interestingly, the roles of individual endothelial surface sugars and lectins in flow-induced responses are function biased; a complexity that makes difficult to identify the exact "mechanosensor(s)" for each response [6,8,[10][11][12][13]15].…”

“…Interestingly, the roles of individual endothelial surface sugars and lectins in flow-induced responses are function biased; a complexity that makes difficult to identify the exact "mechanosensor(s)" for each response [6,8,[10][11][12][13]15]. In contrast, in the case of a defined G-protein coupled receptor, the response triggered by its specific agonist identifies it as the structure of origin, the agonist-sensor [1,3,4,14].…”