Gonzales R A, Crews F T
Department of Pharmacology, Louisiana State University Medical Center, New Orleans 70112.
J Neurochem. 1988 May;50(5):1522-8. doi: 10.1111/j.1471-4159.1988.tb03039.x.
We have shown previously that calcium and guanine nucleotides stimulate the activity of a phosphoinositide (PI) phosphodiesterase in membranes from rat cerebral cortex and that their effects are additive. To understand further guanine nucleotide- and calcium-stimulated PI phosphodiesterase activity, we have investigated the pH sensitivity and effects of inhibitors on the two modes of stimulation. NaF stimulates PI hydrolysis in brain membranes with an EC50 of 2 mM and a maximal effect at 10 mM, suggesting that a guanine nucleotide binding protein can regulate PI phosphodiesterase. Neomycin inhibited guanylylimidodiphosphate (GppNHp)-stimulated PI phosphodiesterase activity in a concentration-dependent manner, with 90% inhibition at 0.3 mM. Neomycin was not as effective at inhibiting calcium-dependent PI hydrolysis (32% inhibition at 0.3 mM). Chloroquine also had a greater inhibitory effect against GppNHp-stimulated PI phosphodiesterase activity compared to calcium-dependent activity. Guanine nucleotide- and NaF-dependent activations of PI phosphodiesterase were strongly pH-dependent, with greatest stimulation observed at pH 5-6 and inhibition at more alkaline pH. Calcium-stimulated PI hydrolysis was not as sensitive to changes in pH and had a peak of activity at pH 9. Our findings of different pH optima and differential sensitivity to inhibitors suggest that calcium and guanine nucleotides may regulate PI phosphodiesterase in rat cortical membranes through independent mechanisms.
我们之前已经表明，钙和鸟嘌呤核苷酸可刺激大鼠大脑皮层膜中磷酸肌醇（PI）磷酸二酯酶的活性，且它们的作用是相加的。为了进一步了解鸟嘌呤核苷酸和钙刺激的PI磷酸二酯酶活性，我们研究了pH敏感性以及抑制剂对这两种刺激模式的影响。NaF刺激脑膜中的PI水解，其EC50为2 mM，在10 mM时达到最大效应，这表明鸟嘌呤核苷酸结合蛋白可以调节PI磷酸二酯酶。新霉素以浓度依赖性方式抑制鸟苷酰亚胺二磷酸（GppNHp）刺激的PI磷酸二酯酶活性，在0.3 mM时抑制率为90%。新霉素在抑制钙依赖性PI水解方面效果不佳（在0.3 mM时抑制率为32%）。与钙依赖性活性相比，氯喹对GppNHp刺激的PI磷酸二酯酶活性也有更大的抑制作用。PI磷酸二酯酶的鸟嘌呤核苷酸依赖性和NaF依赖性激活强烈依赖于pH，在pH 5 - 6时观察到最大刺激，在更碱性的pH时受到抑制。钙刺激的PI水解对pH变化不太敏感，在pH 9时活性达到峰值。我们关于不同pH最佳值和对抑制剂的不同敏感性的研究结果表明，钙和鸟嘌呤核苷酸可能通过独立的机制调节大鼠皮层膜中的PI磷酸二酯酶。
