G protein‐independent stimulation of human myocardial phospholipase C by mastoparan

Abstract

1 Phosphoinositide-speci®c phospholipase C (PLC) is involved in the regulation of many cellular functions. In the myocardium, PLC-generated second messengers play a role in the regulation of contractile function and in the pathophysiology of myocardial hypertrophy. 2 In the present study, the eect of mastoparan, a tetradecapeptide which is capable of activating heterotrimeric G proteins by mimicking the action of an activated receptor, on membrane-bound human myocardial PLC, was investigated in a cell-free assay with exogenous phospholipids as a substrate. 3 Mastoparan stimulated human myocardial PLC approximately two fold with a half-maximal eect at approximately 2 mM and a maximal eect at 10 mM. The peptide did not alter the dependence of PLC on free calcium ions. In order to exclude non-speci®c eects of mastoparan due to its amphiphilic properties, dierent mastoparan derivatives were used as positive and negative controls. Mas17, an inactive mastoparan analogue with phsyical properties very similar to mastoparan, did not induce substantial PLC stimulation in human myocardial membranes. In contrast, Mas7, the most active mastoparan derivative known, caused a more pronounced PLC activation compared with the mother compound indicating that the eect was sequence-speci®c. Human myocardial PLC stimulation was pertussis toxin-insensitive and could not be abolished by addition of excess a-subunits from puri®ed retinal transducin or by excess GDP or GDPbS. In order to investigate whether mastoparan stimulated PLC via pertussis toxin-insensitive a q , a deletion mutant of PLCb 2 de®cient of the site of interaction with a q -subunits was expressed in COS-1 cells. Both wild-type and mutant PLCb 2 were similarly sensitive to stimulation by mastoparan. 4 It is concluded that mastoparan stimulates human myocardial PLC by a mechanism distinct from heterotrimeric G proteins.

Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6

“…The cationic tetradecapeptide mastoparan reportedly activates PLD2 in intact mast cells ( Chahdi et al , 2003 ) and we found that it stimulated PtdBut formation in the PLD-specific transphosphatidylation reaction. However, it did not specifically stimulate PLD in P4E6 and PC3 cell lines because GPEtn and PEtn were the main metabolites released suggesting a preferential activation of PLA 2 and PtdEtn-PLC as observed in other cell types ( Argiolas and Pisano, 1983 ; Schnabel et al , 1997 ; Ferry et al , 2001 ). With benign PNT2C2 cells, mastoparan upregulated basal mechanisms of EtnGP turnover because Etn metabolites were released in the same ratio as from unstimulated cells.…”

In vitroexposure toHymenopteravenom and constituents activates discrete ionotropic pathways in mast cells

“…This is inconsistent with any lytic activity of mastoparan [ 58 ], which could result in large linear currents. Rather, in these cells a classic mastoparan mechanism is more likely, direct activation of Gq [ 59 , 60 ] or PLC [ 61 , 62 ] leading to Ins (1,4,5) P3 production and store release followed by CRAC activation [ 63 , 64 ]. In our bulk assays, we do not detect predicted calcium store release in response to mastoparan, possibly due to the sensitivity of the system.…”

Activation of Gi induces mechanical hyperalgesia poststress or inflammation

“…Of note in this regard, actions of mastoparan have been divided into PTx-sensitive, G i -mediated, and PTx-insensitive, G i - independent signaling pathways. Examples of G i -independent mechanisms include: activation of MAP kinase (Miles et al, 2004), phospholipase C (Nakahata et al, 1990; Schnabel et al, 1997), phospholipase D (Mizuno et al, 1998; Chahdi et al, 2003) and two small molecular weight GTP-biding proteins rho and rac (Koch et al, 1991). Whether one of the previously described G i -independent mechanisms of mastoparan action, or another signaling pathway, mediates the PTx-resistant hyperalgesia induced by mastoparan in the control rats not exposed to prior stress or inflammation remains to be elucidated.…”