RESUMEN
Although it is known that mastoparan, a wasp venom toxin, directly activates Gi/o, mastoparan-induced biological responses are not always explained by this mechanism. For instance, we have demonstrated previously that mastoparan suppressed phosphoinositide hydrolysis induced by carbachol in human astrocytoma cells (FEBS Lett 206:91-94, 1990). In the present study, we examined whether mastoparan affected phosphoinositide hydrolysis by interacting with lipid rafts in PC-12 cells. Mastoparan inhibited UTP-induced increase in [Ca2+]i and phosphoinositide hydrolysis in a concentration-dependent manner. UTP-induced phosphoinositide hydrolysis occurred in lipid rafts, because methyl-beta-cyclodextrin, a disrupting regent of lipid rafts, inhibited the hydrolysis. Mastoparan changed the localization of Galphaq/11 and Gbeta together with cholesterol from lipid rafts to nonraft fractions or cytosol. These changes were inhibited by ganglioside mixtures, suggesting that mastoparan interacts with gangliosides in lipid rafts. In fact, ganglioside mixtures and neuraminidase, but not sialic acid, attenuated the inhibitory effect of mastoparan on phosphoinositide hydrolysis. Furthermore, fluorescence intensity of tyrosine residue of [Tyr3]mastoparan was potentiated by ganglioside mixtures, suggesting the direct binding of mastoparan to gangliosides. Mastoparan caused cytotoxicity of PC-12 cells in a concentration-dependent manner, determined by LDH release. The mastoparan-induced cytotoxicity was significantly inhibited by neuraminidase or gangliosides. The order of inhibitory potency of gangliosides was GT1b approximately GD1b > GD1a > GM1 >> GQ1b, but asialo-GM1 and sialic acid were inactive. These results suggest that mastoparan initially binds to gangliosides in lipid rafts and then it inhibits phosphoinositide hydrolysis by changing the localization of Galphaq/11 and Gbeta in lipid rafts.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Gangliósidos/metabolismo , Microdominios de Membrana/metabolismo , Péptidos/farmacología , Venenos de Avispas/farmacología , Animales , Calcio/metabolismo , Gangliósidos/farmacología , Hidrólisis , Fosfatos de Inositol/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Neuraminidasa/farmacología , Células PC12 , Péptidos/metabolismo , Ratas , Transducción de Señal , Uridina Trifosfato/farmacología , Venenos de Avispas/metabolismoRESUMEN
The ryanodine receptor, a Ca(2+)-releasing channel in sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction coupling of skeletal muscle. In a previous study [Hirata, Nakahata and Ohizumi (2000) Mol. Pharmacol. 57, 1235-1242], we reported that mastoparan caused Ca(2+) release through ryanodine receptor from the heavy fraction of SR (HSR) isolated from rabbit skeletal muscle, and that it specifically bound to a 97 kDa protein which was distinct from Ca(2+)-pump or triadin. The present study was undertaken to identify and characterize the 97 kDa mastoparan-binding protein. The 97 kDa protein was purified from solubilized HSR by DEAE-Sepharose column chromatography and preparative SDS/PAGE. The partial amino acid sequence of the purified 97 kDa protein was matched with that of glycogen phosphorylase (GP). The proteolytic cleavage pattern of the 97 kDa protein was identical with that of GP. Furthermore, [(125)I-Tyr(3)]mastoparan specifically bound to GP. Interestingly, mastoparan-induced Ca(2+) release was inhibited by exogenous addition of GP-a, and mastoparan dissociated GP from HSR. These results indicate that the 97 kDa mastoparan-binding protein is GP, which negatively regulates Ca(2+) release from HSR. There may be a functional cross-talk between Ca(2+) release from HSR and glycogenolysis for energy supply mediated through GP in skeletal muscles.
Asunto(s)
Calcio/metabolismo , Glucógeno Fosforilasa/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Venenos de Avispas/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía en Agarosa , Electroforesis en Gel de Poliacrilamida , Glucógeno Fosforilasa/aislamiento & purificación , Glucógeno Fosforilasa/farmacología , Péptidos y Proteínas de Señalización Intercelular , Radioisótopos de Yodo/metabolismo , Datos de Secuencia Molecular , Músculo Esquelético/química , Músculo Esquelético/efectos de los fármacos , Péptidos , Unión Proteica/efectos de los fármacos , Conejos , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/efectos de los fármacos , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Venenos de Avispas/farmacologíaRESUMEN
The fruit hull of mangosteen, Garcinia mangostana L. has been used as a Thai indigenous medicine for many years. However, its mechanism of action as a medicine has not been elucidated. The present study was undertaken to examine the effects of mangosteen extracts (100% ethanol, 70% ethanol, 40% ethanol and water) on histamine release and prostaglandin E2 synthesis. We found that the 40% ethanol extract of mangosteen inhibited IgE-mediated histamine release from RBL-2H3 cells with greater potency than the water extract of Rubus suavissimus that has been used as an anti-allergy crude drug in Japan. All extracts of mangosteen potently inhibited A23187-induced prostaglandin E2 synthesis in C6 rat glioma cells, while the water extract of Rubus suavissimus had no effect. The 40% ethanol extract of mangosteen inhibited the prostaglandin E2 synthesis in a concentration-dependent manner with relatively lower concentrations than the histamine release. In addition, passive cutaneous anaphylaxis (PCA) reactions in rats were significantly inhibited by this ethanol extract as well as by the water extract of Rubus suavissimus. These results suggest that the 40% ethanol extract of mangosteen has potent inhibitory activities of both histamine release and prostaglandin E2 synthesis.