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In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.

In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [3H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[3H]choline (dipalmitoyl [3H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium deoxycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1-0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1-7.3. Both Ca2+ and Mg2+ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5-1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.

Immunohistochemical localization of pertussis toxin-sensitive GTP-binding proteins in rat cerebellum was investigated using antibodies raised against purified G alpha o and synthetic peptides corresponding to specific sequences of G alpha i1, G alpha i2 and G alpha o was detected mostly in the molecular layer but not in the cell body of the Purkinje cells. G alpha i1 and G alpha i2 were found predominantly in the molecular layer and in addition in the cell body of the Purkinje cells. G alpha i3 was not detected in rat cerebellum. Immunoblot analysis demonstrated that in the cerebellum membrane G alpha 0 was most abundant and surprisingly the amount of G alpha i2 was much higher than that of G alpha i1. Thus, each pertussis toxin substrate was found to be expressed differently in rat cerebellum. The different patterns of expression imply that each subtype of GTP-binding proteins may have a specific function in modulating the neuronal activity in rat cerebellum.

In the present study, we investigated the involvement of protein kinase C (PKC) in antigen (Ag, DNP-Ascaris suum)-induced phospholipase D (PLD) activation of rat peritoneal mast cells. Phorbor myristate acetate (PMA) as well as Ag activated PLD as inferred by phosphatidylethanol (PEt) production. PKC inhibitors, staurosporine and H-7, however, failed to suppress PMA-stimulated PLD activation, suggesting that PLD activation by PMA is independent of PKC. By contrast, Ag-stimulated PLD activity was significantly reduced by staurosporine and slightly by H-7. Surprisingly, the inhibitors inhibited Ag-stimulated phospholipase C (PLC), correlated to the inhibition of PLD. These observations lead us to conclude that in Ag-stimulated mast cells 1,2-diacylglycerol (DG) formed by PLC directly or indirectly stimulates PLD, independently of PKC.

Stimulation by N-formyl-Met-Leu-Phe (fMLP) of rabbit peritoneal neutrophils, in which phosphatidylcholine was preferentially labeled with 1-O-[3H]octadecyl lyso platelet-activating factor, activated phospholipase D, resulting in the formation of [3H]PA from [3H]PC. A direct activator of GTP-binding proteins (G-proteins), NaF, also stimulated [3H]PA formation. fMLP-stimulated [3H]PA formation was inhibited by pertussis toxin (IAP) in a time- and dose-dependent manner. IAP also inhibited fMLP-stimulated IP3 formation, but the inhibition of IP3 formation was significantly greater than that of [3H]PA formation. These results indicate that activation of phospholipase D by fMLP in rabbit neutrophils is mediated by an IAP-sensitive G-protein that may be distinct from a phospholipase C-regulating protein.

As an approach to understanding the molecular basis of the pathophysiology of cerebral ischemia, we examined qualitative and quantitative changes in pertussis toxin substrates, Gi1 and G0, in the membrane of rat cerebral cortex after decapitation. Within 1 min after decapitation, the extent of pertussis toxin-catalyzed [32P]ADP ribosylation of the G proteins in the cerebral cortex membrane was significantly decreased and the magnitude of the decrease became slightly larger upon further incubation of the decapitated brain. Addition of guanine nucleotides, GTP and GDP, or the purified beta gamma subunits of transducin to the membranes of control and ischemic cerebral cortex stimulated [32P]ADP ribosylation of the G proteins. The stimulation of [32P]ADP ribosylation in the control situation by guanine nucleotides was almost to the same extent as that in ischemia. However, the stimulation by transducin beta gamma subunits was different; the control stimulation was greater than that in ischemia. In immunoblots probed with antibodies against Gi1 alpha, G0 alpha, and T beta, the immunoreactivity of the corresponding proteins in ischemia was similar to that in control, suggesting that the amounts of G proteins were not changed in ischemia. These results suggest that ischemia accelerates the dissociation of alpha-GDP-beta gamma to alpha-GDP and free beta gamma and causes the denaturation of the dissociated alpha-GDP, thereby decreasing [32P]ADP ribosylation.

1. Phospholipase B-like activity was found in rabbit brain membranes. 2. The activity was greatly enhanced by 0.025% (w/v) Triton X-100 and was inhibited by both Ca2+ and Mg2+. 3. With increasing pH of the reaction mixture, the activity was augmented. 4. The characteristics of the enzyme activity possibly suggest that phospholipase B in rabbit brain may be distinct from those previously reported.

We compared, by immunoblotting and immunohistochemical staining, the distribution of the alpha subunit of Go in human normal brain tissue with that in human brain tumours of neuroectodermal origin. The ten tumour samples included seven high-grade astrocytomas (grades III and IV) and three low-grade astrocytomas (grade II). Immunoreactivity of high-grade astrocytomas with the alpha subunit of Go (Go alpha) antibody was lower than that of low-grade astrocytomas and normal white matter. In the immunohistochemical study, neoplastic cells in the astrocytomas were found to contain little or no significant Go alpha, although nonlesional white matter was strongly stained with Go alpha antibody. This study suggests that Go alpha antibody may assist in defining the boundary of an astrocytoma, and the difference in Go content may affect the alteration in the physical properties of the membrane, which exhibit, in part, the character of the transformation.

Stimulation by fMet-Leu-Phe analogs of GTPase activity in plasma membranes from rabbit neutrophils was compared with the stimulation of degranulation in intact neutrophils. All four formyl peptides examined (fMet-Leu-Phe-Phe, fMet-Leu-Phe, fNle-Leu-Phe, and fVal-Leu-Phe) were full agonists for both responses. Their ED50 values for the two responses correlated well, although those for GTPase stimulation were uniformly about tenfold greater. The specific antagonist tBoc-Phe-Leu-Phe-Leu-Phe competitively inhibited both GTPase activity and degranulation stimulated by fMet-Leu-Phe; its Ki values were similar for the two responses. Pertussis toxin treatment, in contrast, inhibited the maximal stimulation of both responses by fMet-Leu-Phe with minimal shift in ED50. The inhibitory actions of tBoc-Phe-Leu-Phe-Leu-Phe and pertussis toxin on GTPase activity thus paralleled the effects on degranulation. These observations substantiate the hypothesis that a guanine nucleotide-binding protein that is a pertussis toxin substrate couples the formyl peptide receptors to physiological function in the neutrophil.

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified alpha-subunit of G0 from bovine brain or against synthetic peptides predicted from cDNAs for distinct Gi subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophils. Brain contained both G0 and Gi1. Gi1 was also found to be abundant in heart. All peripheral tissues contained readily detectable amounts of Gi2, whereas only barely detectable amounts of Gi2 were found in brain. Gi3 was found to be prominent in erythrocytes and exists as a minor component of G proteins in neutrophils and liver. Thus, Gi2 appears to be widely disseminated in peripheral rabbit tissues, while other pertussis toxin substrates are more limited in their distribution.

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (Gi). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of Gi. ADP-ribosylation of Gi has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.

A specific, pertussis toxin-sensitive, GTP binding, regulatory protein of neutrophils which we term "Gn" couples the reaction of chemotactic factors with their specific receptors to the resultant stimulation of chemotaxis, granule enzyme secretion, O2- generation, aggregation etc. The ability of four chemotactic formylpeptides to increase the GTPase activity of isolated plasma membranes of rabbit neutrophils correlates almost perfectly with the ability of the same peptides to cause granule enzyme release. These and other findings provide formal evidence for the previous assumption that fMET-Leu-Phe increases the GTPase activity of Gn by reacting with the same receptor that triggers granule enzyme release and other stimulated functions of the neutrophil. The molecular weight of 40 kDa and isoelectric point of 5.5 of the [32P] ADP-ribosylated alpha subunit of Gn, differ slightly but significantly from the corresponding alpha subunits of the other G proteins that are substrates for pertussis toxin. Differences also exist in the patterns of proteolytic fragments of the alpha subunit of Gn and those of the other G proteins. These observations indicate that in neutrophils a G protein distinct from the previously identified pertussis toxin substrates couples the stimulation of chemotactic receptors to the physiological function of cells. The dissociation constants of binding (Kd) for the high and low affinity sites of the formylpeptide receptor of rabbit peritoneal neutrophils and the ability to induce secretion of beta glucosaminidase from the same cells were determined for each of seven chemotactic formylpeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The light-detecting system of retinal rod outer segments is regulated by a guanyl nucleotide binding (G) protein, transducin, which is composed of alpha-, beta-, and gamma-subunits. Transducin couples rhodopsin to the intracellular effector enzyme, a cGMP phosphodiesterase. The beta gamma complex (T beta gamma) is required for the alpha-subunit (T alpha) to interact effectively with the photon receptor rhodopsin. It is not clear, however, whether T beta gamma binds directly to rhodopsin or promotes T alpha binding to rhodopsin only by binding to T alpha. We have found that serum from rabbits immunized with T beta gamma contained a population of antibodies that were reactive against rhodopsin. These antibodies could be separated from T beta gamma antibodies by absorbing the latter on immobilized transducin. Binding of purified rhodopsin antibodies was inhibited by T beta gamma, suggesting that the rhodopsin antibodies and T beta gamma bound to the same site on rhodopsin. We propose that the rhodopsin antibodies act both as antiidiotypic antibodies against the idiotypic T beta gamma antibodies and as antibodies against rhodopsin. This hypothesis is consistent with the conclusion that T beta gamma interacts directly with the receptor. It is probable that in an analogous way, G beta gamma interacts directly with receptors of the adenylate cyclase system.

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, the predominant pertussis toxin substrate in rabbit neutrophils, Gn, was biochemically compared to Gi and Go purified from brain, after being [32P]ADP-ribosylated by activated pertussis toxin and [32P]NAD. On SDS-polyacrylamide gels, a poorly resolved doublet from neutrophil membranes was observed; the upper band, corresponding to approximately equal to 25% labeling, comigrated with Gi-alpha and the predominant lower band, Gn, migrated intermediately between Gi-alpha and Go-alpha. Peptide maps generated by limited-digestion of the labeled Gn, Gi and Go with S. aureus V8 protease were slightly, but definitively and reproducibly different. Isoelectric focusing clearly distinguished Gn from the other two pertussis toxin substrates. The pI value of Gn, 5.60, was distinctly different from those of Gi, 5.75 and 5.80. Although the pI values for Go and Gn were similar (5.60), the patterns of the two proteins were qualitatively different, with Go being resolved into an equal doublet (pI = 5.55 and 5.60) while Gn appeared predominantly as a single band. Thus, Gn is biochemically distinguishable from Gi and Go of brain and these structural differences are most clearly evident following isoelectric focusing.

Evidence is shown that protein kinase C is the major kinase which can phosphorylate histone H-1 in a membrane fraction prepared from rabbit peritoneal neutrophils. Addition of phorbol-12-myristate-13-acetate (PMA) (0.1 microgram/ml) or guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) (10 microM) to the membrane fraction results in an increase of the phosphorylation of histone H-1. To achieve this effect, calcium (20 microM) is required for GTP gamma S but not for PMA. The effect of GTP gamma S, but not PMA is inhibited in membranes obtained from cells pretreated with pertussis toxin. The kinase activity is also enhanced by treatment of the membrane with 10 microM of GppNHp or GTP but not with GDP, GMP, cGMP, ATP, ADP, AMP and cAMP. This is the first direct evidence that a GTP binding protein is involved in the activation of membrane associated protein kinase C.

Hormonal inhibition of adenylate cyclase is mediated by inhibitory receptors and a guanyl nucleotide-binding coupling protein, termed Gi. Similarly, transducin (T), a guanyl nucleotide-binding protein, mediates activation of cGMP phosphodiesterase by the retinal photon receptor, rhodopsin. Gi and T are both heterotrimers consisting of alpha, beta, and gamma subunits; Gi alpha and G beta are similar to T alpha and T beta, respectively. T alpha hydrolyzes GTP in the presence of photolyzed, but not dark, rhodopsin and T beta gamma. Gi alpha and G beta gamma substituted for T alpha and T beta gamma to yield active hybrid complexes, T alpha G beta gamma and Gi alpha T beta gamma. In the absence of T components, rhodopsin-dependent GTPase activity of Gi alpha G beta gamma was observed. Pertussis toxin ADP-ribosylates both T alpha and Gi alpha; ADP-ribosylation of Gi alpha was negligible in the absence of G beta gamma. With G beta gamma, photolyzed, but not dark, rhodopsin unhibited ADP-ribosylation of Gi alpha. In the presence of G beta gamma and photolyzed rhodopsin, GDP and GDP beta S, but not Gpp(NH)p and GTP gamma S, increased the ADP-ribosylation of Gi alpha. The requirements for ADP-ribosylation of Gi alpha by pertussis toxin were similar to those for ADP-ribosylation of T alpha. Rhodopsin appears to interact with Gi in a manner similar to the inhibitory hormone receptors; photolyzed rhodopsin, the active species, corresponds to the agonist-occupied receptor, while dark rhodopsin, the inactive species, can be equated to the free receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of two representative groups of amphiphilic drugs, lysophosphatidylcholine species (myristoyl, palmitoyl and stearoyl) and phenothiazine neuroleptics (promethazine, chlorpromazine and trifluoperazine), on the morphology of intact rabbit platelets, arachidonate release from membrane phospholipids, pseudopod formation and the resulting aggregation of stimulated platelets were examined and compared with those of two known cycloxygenase inhibitors, aspirin and indomethacin. Collagen-induced aggregation was inhibited by relatively low concentrations of amphiphilic drugs in parallel with the prevention of arachidonate release from the membrane phospholipids. Thrombin-and arachidonate-induced aggregations were inhibited by these drugs at higher concentrations, at which they transform intact platelets from discoid to spiny and spherical shape, respectively, and consequently suppress formation of native pseudopods induced by these stimuli. Washing the drug-treated platelets with BSA-Tyrode solution abolished all the effects of drugs. In contrast, the cycloxygenase inhibitors blocked both collagen- and arachidonate-induced aggregations without causing membrane shape change of the intact platelets, although they inhibited thrombin-induced aggregation at extremely high concentrations, at which they did elicit membrane shape change. These observations suggest that these amphiphilic drugs act on the plasma membrane of platelets and impair membrane-linked functions. At lower concentrations they specifically inhibit the arachidonate release from the membrane phospholipids under collagen stimulation; at higher concentrations they drastically perturb the plasma membrane structure, inducing the membrane shape change, and suppressing the native pseudopod formation under thrombin or arachidonate stimulation. The impairment of membrane-linked functions by these amphiphilic drugs is thought to account for their inhibition of stimulus-induced aggregation.

Cepharanthine incorporated into rabbit platelets dose dependently, inhibited calcium influx as well as aggregation in response to collagen, and also inhibited arachidonate release in response to collagen and A23187 in the same concentration ranges. The latter action of cepharanthine was shown not to be due to the direct action on phospholipase A2 molecules but to the depression of susceptibility of substrate phospholipids to enzymatic hydrolysis. These depressed functions, as well as the inhibited aggregation, were almost restored by removing the bound drugs from the platelets. Arachidonate-induced aggregation and prostaglandin synthesis from externally added arachidonate were not suppressed by the addition of cepharanthine. These results suggest that cepharanthine physically changes the lipid properties and thereby inhibits the function of the calcium channel or the susceptibility of substrate phospholipids to enzymatic hydrolysis by phospholipase A2.