ATP-dependent regulation of phospholipase C in permeabilized 3T3 cells

Regulation of phospholipase C (PLC) coupled with a G-protein was studied with Swiss 3T3 cells permeabilized by digitonin. In permeabilized cells, activation of phospholipase C required millimolar concentrations of ATP in addition to a G-protein activator, AlF4- or nonhydrolysable GTP analogues. To determine the mechanism of the action of ATP, we examined the effects of ATP analogues. ATP gamma S directly activated phospholipase C in the presence or absence of AlF4-. On the other hand, neither beta,gamma-methylene ATP nor adenyl-5'-yl imidodiphosphate nor ADP beta S could support the AlF4(-)-dependent activation of phospholipase C. The action of ATP gamma S was not through the substrate supply for phospholipase C, because ATP gamma S did not augment the levels of PIP2 or PIP in permeabilized cells. These results suggested the significance of the gamma-phosphate group of ATP and/or phosphorylation by ATP in the activation of phospholipase C by a putative G-protein.     

Characterization of fMet-Leu-Phe-stimulated phospholipase C in streptolysin-O-permeabilised cells

Phospholipase C (specific for inositol lipids) is known to be present both in membranes and cytosol. Receptor-mediated activation of this enzyme occurs via a guanine nucleotide regulatory protein (G-protein), designated Gp. We have compared the stimulation of this enzyme by fMet-Leu-Phe via the G-protein in HL60 membranes and in permeabilised cells. fMet-Leu-Phe stimulated phospholipase C in membranes at 2 min and the response was dependent on exogenously added GTP. GTP alone also stimulated phospholipase C activity such that at 10 min the response to fMet-Leu-Phe was minimal. In comparison, the response to fMet-Leu-Phe in permeabilised cells was greater in extent but did not require added GTP. However, it was antagonized by GDP analogues (GDP[beta S] greater than GDP greater than dGDP) and by pertussis toxin pretreatment, indicating that fMet-Leu-Phe-stimulated phospholipase C activity was also mediated via Gp. GTP and its analogue GTP[gamma S] also stimulated phospholipase C and their effects were strictly additive to the stimulation obtained with fMet-Leu-Phe. Such additivity was also observed when two receptor-directed agonists, fMet-Leu-Phe and ATP, were used to stimulate intact cells. It is concluded that (a) the size of the response with fMet-Leu-Phe in membranes is limited by the loss of a component, possibly phospholipase C, and (b) stoichiometry and physical organisation of multiple species of G-proteins and/or phospholipases C may explain the independent nature of phospholipase C activation by fMet-Leu-Phe, ATP and guanine nucleotides.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.     

Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.     

A permissive role of pertussis toxin substrate G-protein in P2-purinergic stimulation of phosphoinositide turnover and arachidonate release in FRTL-5 thyroid cells. Cooperative mechanism of signal transduction systems

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.     

Ca2+ inhibits guanine nucleotide-activated phospholipase D in neural-derived NG108-15 cells.

We have investigated the regulation of phospholipase D (PLD) activity by guanine nucleotides and Ca2+ in cells of the NG108-15 neuroblastoma X glioma line that were permeabilized with digitonin. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) caused a nearly sixfold increase (EC50 = 3 microM) in production of [3H]phosphatidylethanol (specific product of the PLD transphosphatidylation reaction). Other GTP analogues were less effective than GTP gamma S, and guanosine-5'-O-(2-thiodiphosphate) inhibited PLD activation by GTP gamma S. Both basal and GTP gamma S-stimulated PLD activities were potentiated by MgATP and Mg2+. Adenosine-5'-O-(3-thiotriphosphate) and ADP also potentiated the effect of GTP gamma S, but non-phosphorylating analogues of ATP had no such effect. The activation of PLD by GTP gamma S did not require Ca2+ and was independent of free Ca2+ ions up to a concentration of 100 nM (resting intracellular concentration). Higher Ca2+ concentrations (greater than or equal to 1 microM) completely inhibited PLD activation by GTP gamma S. It is concluded that elevated intracellular Ca2+ concentrations may negatively modulate PLD activation by a guanine nucleotide-binding protein, thus affecting receptor-PLD coupling in neural-derived cells.     

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils.

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.     

Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.     

Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol

Receptor-linked activation of phospholipase D has been demonstrated recently in a variety of intact cell types including granulocytes, but little is known about the enzyme, its cofactor requirements, and regulation. Using [3H]alkyllysophosphatidylcholine to prelable an endogenous phosphatidylcholine substrate pool in conjunction with transphosphatidylation using ethanol to generate labeled phosphatidylethanol, we demonstrated a novel phospholipase D activity in neutrophil subcellular fractions. Guanosine 5'-O-3-(thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) activated both phosphatidic acid generation and transphosphatidylation. Activity using both activators required the presence of not only plasma membrane but also cytosol, and proteolytic and thermal inactivation demonstrated the requirement for protein factors in both fractions. Using both stimuli, activity increased with increasing cytosol concentration. Product formation was approximately linear for about 10 min with PMA and 30 min with GTP gamma S, and both activators resulted in the total hydrolysis of up to 10% of the labeled phosphatidylcholine. The activity using both activators showed similar broad neutral pH optima, and both required the presence of micromolar levels of calcium, which by itself failed to activate at concentrations up to 1 mM. At low micromolar concentrations of nucleotides, activation was specific for guanine nucleotides and showed a specificity of GTP gamma S greater than guanyl-5'-yl imidodiphosphate greater than GTP, with no effect of GDP and GMP or adenine nucleotides, consistent with the participation of a guanine nucleotide regulatory protein. PMA activation was dependent on the presence of ATP, in particular when dialyzed cytosol was used, and was inhibited by about 50% by staurosporine, supporting a role for protein kinase C. However, purified protein kinase C failed to substitute for cytosol, implicating an additional cytosolic factor(s) in this response. These results indicate that the granulocytic phospholipase D pathway is a complex system that is regulated by at least two activation pathways, each comprised of components in two subcellular compartments.     

Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state.     

A novel pathway for the activation of phospholipase D by P2z purinergic receptors in BAC1.2F5 macrophages.

Macrophages express two distinct types of nucleotide (P2 purinergic) receptors for extracellular ATP: one type induces a Ca(2+)-mobilizing response via the activation of phosphatidylinositol-phospholipase C (PI-PLC) while the second type induces the rapid formation of nonselective pores which are permeated by ions and small (< 1 kDa) organic molecules. We have confirmed the presence of these two ATP receptor types in the BAC1.2F5 murine macrophage cell line and have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) as a selective and potent agonist for the so-called P2z or pore-forming ATP receptor type. Several lines of evidence indicated that occupation of these P2z receptors is also accompanied by a rapid and large increase in the activity of a phosphatidylcholine-selective phospholipase D (PLD) effector enzyme. In cells metabolically labeled with [3H]oleic acid or [3H]glycerol and stimulated in the presence of ethanol, ATP and BzATP induced a severalfold increase in the rate and extent of [3H]phosphatidylethanol (PEt) accumulation. These responses were stimulated only by ATP, BzATP, and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate) with the rank order of potency: BzATP > ATP > ATP gamma A; there was no response to other adenine nucleotides or to non-adenine nucleotides. Significantly, the ability of P2z receptor agonists to stimulate this PLD activity was not dependent on the presence of extracellular [Ca2+] or elevation of cytosolic [Ca2+]. The inability of ionomycin, gramicidin, digitonin, UTP, platelet-activating factor, or phorbol ester to quantitatively mimic these nucleotide effects suggested that activation of this PLD by P2z receptor agonists was not a secondary response due to: 1) enhanced Ca2+ influx; 2) membrane depolarization; 3) nonselective permeabilization of the plasma membrane; 4) stimulation of Ca(2+)-mobilizing ATP receptors; 5) stimulation of a primary PI-PLC pathway; or 6) activation of protein kinase C. These findings suggest that activation of a novel PLD-based signaling pathway may play an important role in the modulation of macrophage function by pore-forming P2z receptors for extracellular ATP.     

Covalent incorporation of 3'-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts.

ATP, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, and several other ATP analogs induced an increase in plasma membrane permeability to monovalent ions and normally impermeant metabolites, including nucleotides, in transformed 3T6 mouse fibroblasts. The rank order of agonist potency for induction of nucleotide channels was BzATP (EC50 = 15 microM) greater than ATP (EC50 = 50 microM) approximately adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) greater than 2-methylthio-ATP (EC50 = 75 microM) approximately 3'-amino-3'-deoxy-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (EC50 = 175 microM). Long wavelength UV illumination of 3T6 cells in the presence of greater than or equal to 20 microM BzATP at 4 degrees C, a nonpermeabilizing temperature, followed by removal of unbound BzATP, resulted in the efflux of 86Rb+ and the release of a prelabeled pool of cytoplasmic nucleotides when the temperature was shifted to 37 degrees C. Photoincorporation of BzATP was inhibited by ATP, ATP alpha S, ATP gamma S, and other ATP analogs that induced an increase in plasma membrane permeability to nucleotides in 3T6 cells under nonphotoactivating conditions. GTP, ITP, UTP, adenosine, and ATP analogs that did not alter plasma membrane permeability to nucleotides under nonphotoactivating conditions also had no effect on BzATP photoincorporation. Photoincorporation of BzATP occurred optimally between pH 6.6 and pH 8.2 but was inhibited at pH 6.0. Photoincorporation of BzATP was also modulated by the osmolarity and the divalent cation concentration of the assay medium. The increase in plasma membrane permeability to nucleotides induced by photoincorporated BzATP occurred at the same rate and had the same temperature, pH, ionic strength, and divalent cation requirements as the increase in plasma membrane permeability to nucleotides induced by ATP and BzATP under nonphotoactivating conditions. These findings support the hypothesis that BzATP can be covalently incorporated into a P2 purinoceptor in 3T6 cells that is coupled to plasma membrane channels for ions and other metabolites.     

Purification and characterization of two G-proteins that activate the beta 1 isozyme of phosphoinositide-specific phospholipase C. Identification as members of the Gq class.

Plasma membranes from bovine liver contain a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity that is activated by guanine nucleotides. The G-proteins involved retained their ability to activate bovine brain PLC-beta 1 in a guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent manner following extraction from the membranes with cholate and reconstitution with phospholipids. This reconstitution assay was used to purify the G-proteins by chromatography on heparin-Sepharose, DEAE-Sephacel, octyl-Sepharose, hydroxylapatite, Mono Q, and Sephacryl S-300 gel filtration. Gel electrophoresis showed that two alpha-subunits with molecular mass of 42 and 43 kDa were isolated to a high degree of purity, together with a beta-subunit. Neither alpha-subunit was a substrate for pertussis toxin-catalyzed ADP-ribosylation. Gel filtration of the final activity indicated an apparent molecular mass of 95 kDa, suggesting the presence of an alpha beta gamma heterotrimer. Immunological data revealed that the 42- and 43-kDa proteins were related to alpha-subunits of the Gq class recently purified from brain (Pang, I.-H., and Sternweis, P. C. (1990) J. Biol. Chem. 265, 18707-18712) and identified by molecular cloning (Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117). The activation of PLC-beta 1 by the purified G-protein preparation was specific for nonhydrolyzable guanine nucleotides, the efficacy decreasing in order GTP gamma S greater than guanylimidodiphosphate greater than guanylyl(beta,gamma-methylene)-diphosphonate. Half-maximal activation required 4 microM GTP gamma S suggesting that the affinity of the G-proteins for GTP analogues is low. The GTP gamma S-dependent activation of PLC-beta 1 required millimolar Mg2+ and was inhibited by guanosine 5'-O-(2-thiodiphosphate) and by excess beta gamma-subunits. Aluminum fluoride also activated PLC-beta 1 in the presence of the G-proteins. The G-proteins were inactive toward PLC-gamma 1 or PLC-delta 1. In summary, these findings identify two G-protein activators of PLC-beta 1 that have the properties of heterotrimeric G-proteins and are members of the Gq class.     

Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells.

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.     

Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system.

Atrial natriuretic peptide (ANP) binds to a transmembrane receptor having intrinsic guanylyl cyclase activity; this receptor has been designated GC-A. Binding of ANP to GC-A stimulates its catalytic activity, resulting in increased production of the second messenger, cyclic GMP. Here we show that GC-A can be expressed in insect cells using a recombinant baculovirus and that the expressed protein retained its abilities to bind ANP and to function as an ANP-activated guanylyl cyclase. In addition, GC-A produced in insect cells was absolutely dependent on the presence of adenine nucleotides for activation by ANP. Millimolar concentrations of ATP were required for optimal activation. The relative potencies of various nucleotides for activation was adenosine 5'-O-(thiotriphosphate) greater than ATP greater than ADP, adenosine 5'-(beta, gamma-imino)triphosphate greater than ADP beta S. AMP had no effect. These studies suggest that binding of an adenine nucleotide, most likely to the protein kinase-like domain of GC-A, is absolutely required for ANP activation. Regulation of guanylyl cyclase activation by adenine nucleotides represents a novel mechanism for the modulation of signal transduction, possibly analogous in some respects to the role of guanine nucleotides and G proteins in the regulation of adenylyl cyclase activity.     

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.     

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.