RESUMEN
Intracellular calcium [Ca(2+)](i) is mobilized in many cell types in response to activation of phosphoinositide (PIP(n)) signaling pathways involving PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3). To further explore the relationship between increases in intracellular PIP(n) concentrations and mobilization of [Ca(2+)](i), each of the seven phosphorylated phosphoinositides (PIP(n)s) were delivered into cells and the metabolism and physiological effects of the exogenously administered PIP(n)s were determined. The efficient cellular delivery of fluorophore-tagged and native PIP(n)s was accomplished using histone protein, neomycin, and dendrimeric polyamines. PtdIns(4,5)P(2) fluorophore-tagged analogs with short- and long-acyl chains were substrates for cellular enzymes in vitro and for phospholipases in stimulated fibroblasts. PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2), each induced calcium mobilization rapidly after exogenous addition to fibroblasts. PtdIns(3,4,5)P(3) induced a significant, but smaller increase in intracellular calcium. These observations suggest that PIP(n)s other than PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) may have direct roles in signaling involving [Ca(2+)](i).
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Líquido Intracelular/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de Señal/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Colorantes Fluorescentes , Histonas/metabolismo , Histonas/farmacología , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/farmacología , Ratones , Células 3T3 NIH , Fosfatidilinositoles/química , Fosfatidilinositoles/farmacología , Fosforilación , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
A fluorogenic analog of the PLA(2) substrate PC, named Dabcyl-BODIPY-PC, or simply DBPC, was synthesized with a fluorescence quencher (Dabcyl, 4-[(4-[N,N-dimethylamino]phenyl)azo]benzoic acid) in the sn-1 acyl chain and a BODIPY fluor in the sn-2 acyl chain. DBPC was recognized by sPLA(2) from each of the four sources examined (bee venom, human synovial fluid, cobra venom, and bovine pancreas). A dramatic and quantifiable fluorescence enhancement of DBPC occurred upon phospholipase digestion both in the presence and absence of excess PC. Both real-time and endpoint assays for PLA(2) were sensitive, consistent, and rapid. Thus, DBPC can be used as a sensitive fluorogenic probe for in vitro high-throughput screening assays for PLA(2) activation and inhibition and would expedite studies of PLA(2) in cellular signaling, in vitro screening for drug discovery, and subcellular localization of enzyme activity.