Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

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 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.     

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.     

32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.     

A second guanyl nucleotide-binding site associated with adenylate cyclase. Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin

There are two functionally and physically distinct types of guanyl nucleotide site associated with the adenylate cyclase system of pigeon erythrocytes. One is on the well known regulatory protein, N, that mediates the adenylate cyclase response to hormones, guanyl nucleotides and fluoride, and is the substrate for ADP-ribosylation by cholera toxin. We now describe a second site that must be occupied by GTP or an analog of GTP before N can be ADP-ribosylated. We call this second site S. It differs from the site on N in many respects. GTP appears to be rapidly hydrolyzed when it is bound to N but not when bound at S. GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bind stably to both sites but the binding of GTP gamma S to N is more sensitive to EDTA and is more easily prevented by guanosine 5'-O-(2-thiodiphosphate). The nucleotide binding only to S is promoted by the cytosolic protein required by cholera toxin. Isoproterenol decreases GTP gamma S binding to S while indirectly increasing GTP gamma S binding to N. By adjusting the binding conditions, the nucleotides bound functionally to N and S can be varied independently and then the effect of ADP-ribosylation upon the adenylate cyclase activity can be seen to depend on the type of nucleotide bound to N. This activity rises, falls slightly, or remains at zero, if N is occupied by GTP, GTP gamma S, or guanosine 5'-O-(2-thiodiphosphate, respectively.     

Kinetics of "P"-site-mediated inhibition of adenylyl cyclase and the requirements for substrate

The kinetics of "P"-site-mediated inhibition of adenylyl cyclase was studied with the detergent-solubilized enzyme from rat brain. Mn2(+)-activated adenylyl cyclase exhibited typical noncompetitive inhibition by 2'-d3'-AMP or 2',5'-dideoxyadenosine (2',5'-ddAdo). However, enzyme that was preactivated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or proteolytically with ninhibin (+ GTP gamma S) exhibited apparently uncompetitive inhibition with either 2'-d3'-AMP or 2',5'-ddAdo and with either MgATP or MgApp(NH)p (adenosine 5'-(beta gamma-imino)triphosphate) as substrate. Inhibition increased with increasing substrate concentration, consistent with distinct domains for catalysis and the P-site and the formation of a 2'-d3'-AMP.C.MgATP complex. This conclusion was supported by the kinetics of product inhibition. For both cAMP and inorganic pyrophosphate (MgPPi) inhibition was mixed, suggesting that product release is likely random sequential. Although MgPPi enhanced inhibition in the presence of P-site agonist, it did not affect the dissociation constant for P-site agonist. The uncompetitive character of P-site-mediated inhibition and the independence of inhibition by MgPPi and P-site agonist imply that the P-site binding domain is distinct from the substrate binding domain. Given the structural requirements for catalysis and for P-site-mediated inhibition, these domains would be expected to be homologous. Sensitivity to P-site-mediated inhibition was also dependent on the structure of ATP, with the following IC50 values for 2'-d3'-AMP: ATP approximately 2'-dATP (approximately 1 microM); adenosine 5'-O-(3-thiotriphosphate) (approximately 5 microM); App(NH)p (approximately 30 microM); adenosine 5'-(beta gamma-methylene)triphosphate (approximately 300 microM). The differing effectiveness of the ATP analogs to support P-site inhibition was not due to their binding at the P-site. This effect of substrate was also observed with the platelet enzyme and was independent of the means by which the enzyme was activated, whether by Mn2+ or proteolytically by ninhibin/GTP gamma S, suggesting it is a general characteristic of P-site-mediated inhibition. The data suggest a structure for activated adenylyl cyclase such that one nucleotide binding domain, selective for ATP vis-à-vis other ATP analogs, allosterically modulates a proximate P-site domain.     

Synthesis of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)guanosine 5'-triphosphate and a study of its inhibitory properties with adenylate cyclase

A fluorescent GTP analog 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) guanosine 5'-triphosphate (TNP-GTP) has been prepared and some of its physical properties characterized. TNP-GTP was found to be a potent inhibitor of chick embryo heart adenylate cyclase as activated by guanyl 5'-(beta,gamma-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 microM range. It also appeared to inhibit substantially basal adenylate cyclase in this system. TNP-GTP demonstrated an effective competition with [3H]GppNHp, binding to membranes equivalently to GppNHp and about three times better than GTP. 8-Azidoguanosine 5'-triphosphate (8N3GTP) mimics GTP activation of chick embryo heart adenylate cyclase and [gamma-32P]8N3GTP is effectively photoincorporated into a 42,000- to 44,000-Mr doublet when proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TNP-GTP effectively prevents this photoincorporation, as does GTP, at concentrations that agree with their respective apparent inhibition and activation binding constants. The data suggest that TNP-GTP could prove to be a valuable tool for studying the mechanisms of GTP regulation of adenylate cyclase and other GTP-regulated systems.     

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.     

Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis

Provision of GTP (or other nucleotides capable of acting as ligands for activation of G-proteins) together with Ca2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with Gp, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3H]inositol to test whether the requirement for GTP in exocytosis is an expression of Gp activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP- gamma-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-gamma-S in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C.     

Roles of GTP and GDP in the regulation of the thyroid adenylate cyclase system

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regeneratign system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP and GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of nucleotide-regenerating system, addition of GDP to the adenylate cyclase assay mixture int he parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparatiosn possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrst to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic componenet of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Metabolism of arachidonic acid by isolated rat hepatocytes, renal cells and by some rabbit tissues. Detection of vicinal diols by mass fragmentography

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regenerating system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP or GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of a nucleotide-regeneration system, addition of GDP to the adenylate cyclase assay mixture resulted in the parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparations possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrast to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic component of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

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.     

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.     

Detergent-induced distinctions between fluoride- and vanadate-stimulated adenylate cyclases and their responses to guanine nucleotides

The influence of detergents on fluoride- and vanadate-stimulated adenylate cyclases was investigated with enzyme from liver and adipocyte plasma membranes. Stimulation of the adipocyte cyclase by Na3VO4 was maximal (sixfold) at 3 mM, was not additive with fluoride stimulation, and was readily reversed by washing of the membranes. Vanadate stimulation of the hepatic cyclase was specifically blocked by catechol, which had no effect on basal activity or on fluoride- or glucagon-stimulated activities. The hepatic enzyme, stimulated by fluoride ion, guanyl-5'-yl-(beta,gamma-imino)diphosphate (GPP(NH)P), or GPP(NH)P and glucagon, was inhibited by vanadate with 50% inhibition seen with 2 to 6 mM vanadate. The fluoride-activated adipocyte adenylate cyclase was inhibited by guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) more potently than by GPP(NH)P, with 50% inhibition being seen with 10 nM GTP gamma S or 100 nM GPP(NH)P. These nucleotides also inhibited the vanadate-stimulated enzyme, but with one-third the potency seen with the fluoride-activated cyclase. Dispersion of the adipocyte cyclase by Lubrol-PX into a 30,000g supernatant fraction caused no change in activation of the enzyme by fluoride, but reduced vanadate-stimulated activity 80%. By comparison, this treatment enhanced stimulation by GPP(NH)P twofold and by GTP gamma S threefold. More importantly, perhaps, the treatment with detergent blocked inhibition of the basal enzyme by GTP, blocked inhibition of fluoride- and vanadate-stimulated cyclases by GTP, GPP(NH)P, or GTP gamma S, and rendered vanadate-stimulated activity sensitive to enhancement by guanine nucleotides. The data indicate differences in the actions of vanadate and fluoride, made evident by the influence of guanine nucleotides and detergent treatment. The observations would be consistent with the idea that the effects of vandate may be due to the formation of GDP X V on the enzyme. The data strongly suggest that treatment of adenylate cyclase with Lubrol-PX causes a functional blockade in the guanine nucleotide-dependent inhibitory regulation (mediated by Ni), thereby allowing activation by the stimulatory guanine nucleotide-dependent regulatory component (Ns).     

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.     

Synergistic inhibition of human platelet adenylate cyclase by stable GTP analogs and epinephrine

Adenylate cyclase inhibition by stable GTP analogs and their interaction with epinephrine were studied in human platelet membranes. Whereas basal enzyme activity was increased by these nucleotides, the stable GTP analogs decreased the adenylate cyclase activity stimulated by fluoride or forskolin by maximally 60 to 70%, with the potency order, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanyl-5'-ylimidodiphosphate greater than guanyl-5'-ylmethylenediphosphate. The inhibition of the forskolin-stimulated enzyme by GTP gamma S was half-maximal at about 4 nM, occurred after a time lag period, which was inversely related to the GTP gamma S concentration, and was resistant to washing of the membranes. Prostaglandin E1-stimulated activity exhibited a biphasic response towards GTP gamma S, with activation occurring at low (1 nM) and inhibition at higher GTP gamma S concentrations. The inhibitory effect of GTP gamma S was competitively antagonized by GTP. This antagonism was prevented by epinephrine, which inhibited the stimulated platelet adenylate cyclase in the presence of GTP to the same degree as observed with GTP gamma S alone. In the absence of GTP, epinephrine largely diminished the time lag required for the inhibitory action of GTP gamma S. Furthermore, the decrease in final activity induced by GTP gamma S was amplified by epinephrine. Whereas the acceleration of the inhibitory action of GTP gamma S was observed at low and high GTP gamma S concentrations, the amplification by epinephrine was observed only at submaximally effective concentrations of GTP gamma S.     

Dual effects of ATP on phosphatidylinositol breakdown in rat hepatocyte membranes

The mechanisms whereby adenosine-5−triphosphate (ATP)_regulated the inositol phospholipid-signalling system were studied in rat hepatocytes. Intact hepatocytes respond to extracellular ATP, adenosine-5′-O-(3-thiotriphosphate) (ATPγS), ADP and weakly to guanosine-5′-triphosphate (GTP), but not to other purine nucleotides (GDP or AMP). This is consistent with the ideal that a P₂ purinergic receptor is coupled to the phosphatidylinositol metabolism in these cells. Partially purified plasma membranes prepared from myo-[³H]inositol prelabelled hepatocytes exhibit a phosphatidylinositol-4,5-bisphosphate phospholipase C activity sensitive to ATP, ATPγS and guanosine-5′-O-(3-thiotriphosphate) (GTPγS). Moreover the GTPγS effect of greatly enhanced by ATP and ATPγS. These potentiating effects differ according to the adenylnucleotide considered. ATP produces (1) an increase in the GTPγS-PLC sensitivity, (2) a potentiation of the phospholipase C (PLC) response induced by maximal dose of GTPγS, and (3) an increase in the inositol lipids pools. At variance, ATPγS, a nonhydrolysable analogue of ATP, only increases the PLC-sensitivity towards GTPγS. These results may signify that ATP stimulates inositol phosphate accumulation via at least two distinct mechanisms (i) a direct activation of a P₂ purinergic receptor coupled to a PLC via a GTP binding protein and (ii) a stimulation of the phosphatidylinositol (PI) and phosphatidyinositol-4-phosphate (PIP) kinases which increased the pool of phospholipase C substrates.     

Ethanol does not stimulate guanine nucleotide-induced activation of phospholipase C in permeabilized hepatocytes

Guanine nucleotides are thought to mediate the interaction of the receptors for calcium-mobilizing hormones and phosphoinositide-specific phospholipase C. In the present study the characteristics of guanine nucleotide-dependent phospholipase C activation were studied in [3H]inositol-labeled permeabilized hepatocytes. The nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanyl-5'-yl imidodiphosphate stimulated the production of inositol phosphates by phospholipase C. The effect was concentration-dependent with half-maximal and maximal stimulation occurring with 0.6 and 10 microM GTP gamma S, respectively. The guanine nucleotide-induced stimulation of phosphoinositide breakdown was selective for phosphatidylinositol (4,5)-bisphosphate over phosphatidylinositol (4)-phosphate. The individual inositol phosphates formed after maximal GTP gamma S exposure were analyzed by high-performance liquid chromatography. Inositol 1,4,5-trisphosphate was rapidly produced, followed by the formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate. Ethanol is known to activate hormone-sensitive phospholipase C in intact rat hepatocytes. Ethanol (0.3 M) was ineffective in altering the characteristics of GTP gamma S-stimulated phospholipase C activation, in both digitonin-treated and sonicated hepatocytes. The metabolism of the various inositol phosphate isomers was unaffected by ethanol. The findings demonstrate the potential for the use of permeabilized hepatocytes in the analysis of phospholipase C activation by guanine nucleotides. Ethanol does not activate phospholipase C by altering this process.