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Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx.

ABSTRACT: The role of bivalent cations in ATP-stimulated phospholipase D (PLD) activity was investigated in human leukaemic lymphocytes. Cells were labelled with [3H]oleic acid and incubated with extracellular ATP or benzoylbenzoic ATP in the presence of 1 mM Ca2+ and butanol, and PLD activity was assayed by the accumulation of [3H]phosphatidylbutanol ([3H]PBut). ATP stimulated PLD activity in a dose-dependent manner, and the inhibitory effects of suramin, oxidized ATP and extracellular Mg2+ suggested that the effect of ATP was mediated by P2Z purinoceptors known to be present on lymphocytes. Thapsigargin increased cytosolic [Ca2+] but did not stimulate PLD activity, whereas preloading cells with a Ca2+ chelator reduced cytosolic [Ca2+] and, paradoxically, potentiated ATP-stimulated [3H]PBut accumulation. ATP-stimulated [3H]PBut formation was supported by both Ba2+ and Sr2+ when they were substituted for extracellular Ca2+. Addition of EGTA to block bivalent cation influx inhibited the majority of ATP-stimulated PLD activity. Furthermore ATP-stimulated PLD activity showed a linear relationship to extracellular [Ba2+], and ATP-induced 133Ba2+ influx also had a linear dependence on extracellular [Ba2+]. These results suggest that ATP stimulates PLD activity in direct proportion to the influx of bivalent cations through the P2Z-purinoceptor ion channel and that this PLD activity is insensitive to changes in bulk cytosolic [Ca2+].

SUBMITTER: Gargett CE

PROVIDER: S-EPMC1216939 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Premenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats. METHODS:To test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750??g/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1? and ENaC?) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000?nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors. RESULTS:Acute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3. CONCLUSION:These data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Project description:Phospholipase C-eta2 (PLC-eta2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667-676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128-29134]. In this study, we investigated the direct regulation of PLC-eta2 by Gbetagamma subunits of heterotrimeric G proteins. Coexpression of PLC-eta2 with Gbeta 1gamma 2, as well as with certain other Gbetagamma dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gbeta 1gamma 2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-eta2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-eta2 and its potential direct activation by Gbetagamma, a construct of PLC-eta2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-eta2 was quantified after reconstitution with PtdIns(4,5)P 2-containing phospholipid vesicles, and values for K m (14.4 microM) and V max [12.6 micromol min (-1) (mg of protein) (-1)] were similar to activities previously observed with purified PLC-beta or PLC-epsilon isozymes. Moreover, purified Gbeta 1gamma 2 stimulated the activity of purified PLC-eta2 in a concentration-dependent manner similar to that observed with purified PLC-beta2. Activation was dependent on the presence of free Gbeta 1gamma 2 since its sequestration in the presence of Galpha i1 or GRK2-ct reversed Gbeta 1gamma 2-promoted activation. The PH domain of PLC-eta2 is not required for Gbeta 1gamma 2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gbeta 1gamma 2. Taken together, these studies illustrate that PLC-eta2 is a direct downstream effector of Gbetagamma and, therefore, of receptor-activated heterotrimeric G proteins.

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Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx.

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