Okajima F, Sato K, Nazarea M, Sho K, Kondo Y
Department of Physical Biochemistry, Gunma University, Maebashi, Japan.
J Biol Chem. 1989 Aug 5;264(22):13029-37.
Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an "inhibitory action" and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as "stimulatory actions" in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the "inhibitory actions" of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.
在FRTL-5细胞中，发现细胞外ATP和其他嘌呤能激动剂通过抑制腺苷酸环化酶来抑制cAMP积累，此为“抑制作用”，和/或与磷脂酶C或A2激活及Ca2+动员相关联刺激花生四烯酸释放，此为“刺激作用”。以ATP为代表的一组P2-激动剂的刺激作用，即使在约41-kDa膜蛋白完全被ADP-核糖基化后，用胰岛激活蛋白（IAP）、百日咳毒素预处理细胞也只能部分抑制。只有刺激作用中对IAP敏感的部分能被腺苷拮抗剂1,3-二乙基-8-苯基黄嘌呤（DPX）拮抗。GTP和8-溴腺苷5'-三磷酸（Br-ATP）浓度比ATP高两到三个数量级时也发挥刺激作用，尽管它们对IAP和DPX都完全不敏感。用[35S]ATPγS和[3H]DPX进行的配体结合实验表明，ATP占据DPX敏感和不敏感的受体位点，而GTP只占据可被ATP取代的DPX不敏感位点。因此，GTP作用对DPX不敏感与其无法占据DPX敏感位点有关。腺苷5'-O-(1-硫代三磷酸)（ATPαS）、腺苷5'-O-(2-硫代二磷酸)（ADPβS）以及P1-激动剂如AMP和N6-(L-2-苯基异丙基)-腺苷（PIA）未表现出任何刺激作用。然而，这些激动剂显著增强了GTP或Br-ATP的刺激作用。PIA等的这种允许作用对IAP和DPX都敏感，ATP刺激作用的一部分以及PIA和ATP的“抑制作用”也是如此。我们得出结论，介导嘌呤能激动剂通过DPX敏感嘌呤能受体产生抑制作用的IAP底物G蛋白可能不直接与磷脂酶C或A2系统相连，而是以间接或允许的方式增强与DPX不敏感P2受体相连的系统。
