Messenger molecules derived from membrane lipids.

Significant advances have been made recently concerning mechanisms involved in the regulation of cell calcium levels. The mechanisms and physiological significance of agonist-induced phosphatidylcholine hydrolysis are also becoming clearer.

Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts.

alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.

A region of adenylyl cyclase 2 critical for regulation by G protein beta gamma subunits.

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.

Inhibitory effect of epinephrine on insulin-stimulated glucose uptake by rat skeletal muscle.

The effect of epinephrine on basal and insulin-stimulated glucose uptake in perfused hindlimbs of fed rats was studied. Insulin increased glucose uptake in a dose-dependent manner from a basal value of 1.5+/-0.3 up to a maximum value of 5.3+/-0.9 mumol/min per 100 g with 6 nM (1 m U/ml). Epinephrine at 10 nM and 0.1 muM also increased glucose uptake to 2.6+/-0.1 and 3.1+/-0.1 mumol/min per 100 g, respectively. These same concentrations of epinephrine, however, suppressed the insulin-stimulated glucose uptake to 3.2+/-0.3 mumol/min per 100 g. Both the stimulatory and inhibitory effects of epinephrine on glucose uptake were completely reversed by propranolol, but were not significantly altered by phentolamine. Uptake of 3-O-methylglucose and 2-deoxyglucose into thigh muscles of the perfused hindlimbs was stimulated fivefold by insulin, but was unaffected by epinephrine. Epinephrine also did not inhibit the stimulation of uptake by insulin. Epinephrine decreased the phosphorylation of 2-deoxyglucose, however, and caused the intracellular accumulation of free glucose. These last two effects were more prominent in the presence of insulin. Whereas epinephrine caused large rises in glucose-6-P and fructose-6-P, insulin did not alter the concentration of these metabolites either in the absence or presence of epinephrine.THESE DATA INDICATE THAT: (a) epinephrine has a stimulatory effect on glucose uptake by perfused rat hindlimbs that does not appear to be exerted on skeletal muscle; (b) epinephrine does not affect hexose transport in skeletal muscle; (c) epinephrine inhibits insulin-stimulated glucose uptake in skeletal muscle by inhibiting glucose phosphorylation. It is hypothesized that the inhibition of glucose phosphorylation is due to the stimulation of glycogenolysis, which leads to the accumulation of hexose phosphates, which inhibit hexokinase.

Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.

Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes.

Bacterial toxins affect early events of T lymphocyte activation.

The effects of pertussis toxin and cholera toxin on early events of T lymphocyte activation were examined in the T lymphocyte cell line, Jurkat. Pertussis toxin treatment of these T cells increased inositol phosphates production and led to increases in intracellular free calcium concentration. These effects were produced by the isolated B (binding) subunit of pertussis toxin, alone. Inositol phosphates production resulting from perturbation of the T cell antigen receptor-CD3 complex by MAb was not affected by pertussis toxin treatment but was markedly inhibited by cholera toxin. This effect of cholera toxin paralleled elevations in cAMP content. However, forskolin, in concentrations equipotent for cAMP production, was a weaker inhibitor of inositol phosphates production. Cholera toxin inhibition of inositol phosphates production did not result from inhibition of baseline incorporation of inositol into phosphoinositide substrates of phospholipase C. These studies underline the complexity of toxin effects on cellular systems and suggest that other approaches will be required to implicate guanine nucleotide-binding regulatory proteins in control of the early events of T lymphocyte activation. However, the data presented here provide a molecular basis for the clinical observations of lymphocytosis and the in vitro observations of lymphocyte mitogenesis after pertussis toxin stimulation.

Phospholipase D activity is required for actin stress fiber formation in fibroblasts.

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ~50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of alpha-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by alpha-actinin.

Phosphatidylcholine breakdown and signal transduction.

PC hydrolysis by PLA2, PLC or PLD is a widespread response elicited by most growth factors, cytokines, neurotransmitters, hormones and other extracellular signals. The mechanisms can involve G-proteins, PKC, Ca2+ and tyrosine kinase activities. Although an agonist-responsive cytosolic PLA2 has been purified, cloned and sequenced, the agonist-responsive form(s) of PC-PLC has not been identified and no form of PC-PLD has been purified or cloned. Regulation of PLA2 by Ca2+ and MAPK is well established and involves membrane translocation and phosphorylation, respectively. PKC regulation of the enzyme in intact cells is probably mediated by MAPK. The question of G-protein control of PLA2 remains controversial since the nature of the G-protein is unknown and it is not established that its interaction with the enzyme is direct or not. Growth factor regulation of PLA2 involves tyrosine kinase activity, but not necessarily PKC. It may be mediated by MAPK. The physiological significance of PLA2 activation is undoubtedly related to the release of AA for eicosanoid production, but the LPC formed may have actions also. There is much evidence that PKC regulates PC-PLC and PC-PLD and this is probably a major mechanism by which agonists that promote PI hydrolysis secondarily activate PC hydrolysis. Since no agonist-responsive forms of either phospholipase have been isolated, it is not clear that PKC exerts its effects directly on the enzymes. Although it is assumed that a phosphorylation mechanism is involved, this may not be the case, and regulation may be by protein-protein interactions. G-protein control of PC-PLD is well-established, although, again, it has not been demonstrated that this is direct, and the nature of the G-protein(s) involved is unknown. In some cell types, there is evidence of the participation of a soluble protein, which may be a low Mr GTP-binding protein. What role this plays in the activation of PC-PLD is obscure. Agonist activation of PC hydrolysis in cells is usually Ca(2+)-dependent, but the step at which Ca2+ is involved is unclear, since PC-PLD and PC-PLC per se are not influenced by physiological concentrations of the ion. Most growth factors promote PC hydrolysis and this is mainly due to activation of PKC as a result of PI breakdown. However, in some cases, PC breakdown occurs in the absence of PI hydrolysis, implying another mechanism that does not involve PI-derived DAG.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of phospholipase D.

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H(2)O(2) treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol.

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1, 2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase alpha and cytosolic Ca2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase alpha or between the changes in cytosolic Ca2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca2+ and activation of glycogen phosphorylase. Addition of an alpha1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca2+ and phosphorylase alpha, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca2+-mobilizing hormones regulate phosphorylase a through a Ca2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.

Phospholipid interactions affect substrate hydrolysis by bovine brain phospholipase A1.

The specificity of substrate hydrolysis by bovine brain phospholipase A1 (PLA1) was examined. In the presence of Mg2+, using pH values of 7 to 9, the purified enzyme deacylated 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine yielding 2-[1-14C]arachidonoyl-lysophosphatidylethanolamine at a rate of 70 mumol/min per mg. In the absence of Mg2+, however, the reaction rate slowed at pH values above 7.25. In contrast, brain PLA1 slowly (3.8 mumol/min per mg) hydrolyzed 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine (PAPC) unless phosphatidylserine (PS) was included. Maximal PAPC hydrolyzing activity required a PAPC/PS molar ratio of 2.5:1, Mg2+, and a pH value of 8.5-9.5. Replacing PS with phosphatidylethanolamine (PE) or phosphatidic acid (PA), but not phosphatidylinositol (PI), produced a similar effect. Moreover, hydrolysis of either arachidonoyl-substituted or dipalmitoyl-substituted PC at pH 7.5 was enhanced by increasing the mol fraction of PE. Brain PLA1 also hydrolyzed 1-stearoyl-[1-14C]arachidonoyl-PI with high velocity, but only if the substrate was dispersed in PE vesicles. In contrast, the velocity of PS, 1-palmitoyl-lyso-PC or diacylglycerol hydrolysis was low and unaffected by PE. In summary, PLA1 hydrolyzed PE with high velocity and specificity, whereas a high rate of PC or PI hydrolysis was observed only if PS, PE, or PA was present. In addition, PLA1 activity was greatly influenced by pH and Mg2+, implying that the substrate conformation is important to the catalytic efficiency of PLA1. Finally, the high rate of PE, PC or PI hydrolysis suggests PLA1 significantly contributes to the turnover of these phospholipids in the brain.

Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells.

Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity. In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates and inactivates Rho proteins. Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists. Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial. GTPgammaS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment. Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPgammaS on PLD. ATP produced a significant enhancement of the in vitro effect of GTPgammaS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases. However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP2.

Multiple phosphorylation of rat-liver glycogen synthase by protein kinases.

The phosphorylation sites in liver synthase were studied using gel filtration and high performance liquid chromatography of 32P-labeled tryptic peptides. Phosphorylase b kinase, calmodulin-dependent glycogen synthase kinase and glycogen synthase kinase 4 from liver phosphorylated the same low Mr tryptic peptide. cAMP-dependent protein kinase mainly phosphorylated the low Mr tryptic peptide, but also incorporated phosphate into two other peptides. Glycogen synthase kinase 5 phosphorylated a single tryptic peptide, whereas glycogen synthase kinase 3 phosphorylated several tryptic peptides. Calcium-phospholipid-dependent protein kinase phosphorylated two tryptic peptides, the major one of which had the same chromatographic properties as the low Mr peptide described above. These findings confirm that liver glycogen synthase undergoes multi-site phosphorylation and suggest that the topography of the sites is generally similar to that in muscle glycogen synthase.

Calcium regulation of guanine nucleotide activation of hepatic adenylate cyclase.

Pretreatment of isolated rat liver plasma membranes by washing with NaHCO3 buffer or by exposure to the chelator ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5'-(beta-gamma-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca2+, Mg2+ or Mn2+. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca2+. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca2+ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P. Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca2+-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca2+. The effects of low concentrations (less than 100 microM) of the phenothiazine could be reversed totally by Ca2+ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca2+ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.

Purification of the hepatic vasopressin receptor using a novel affinity column.

A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5'-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms.

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.

Alterations in vasopressin and angiotensin II receptors and responses during culture of rat liver cells.

Vasopressin and angiotensin II binding and responses were studied in hepatocytes in primary culture for 4 h and 24 h. After 24 h of culture, angiotensin II was completely ineffective in elevating cytosolic [Ca2+], whereas the maximum [Ca2+] response to vasopressin was decreased by 66% and the sensitivity to the hormone was decreased approx. 20-fold compared with values after 4 h of culture. The dissociation constant (KD) for vasopressin binding to the cells was not significantly changed during 24 h of culture, but the Bmax was decreased by 63% compared with 4 h of culture. There was also no change in the KD for angiotensin II binding from 4 h to 24 h, but the Bmax was decreased by 90%. After 24 h of culture, there was no change in the plasma membrane concentration of phosphatidylinositol 4,5-bisphosphate or in the basal cell concentration of inositol trisphosphate. However, the trisphosphate did not increase with 100 nM angiotensin II and the response to 100 nM vasopressin was reduced by 66% compared with that at 4 h. The effect of guanosine 5'-(3-O-thiol) triphosphate on the polyphosphoinositide phospholipase C activity of liver cell plasma membranes was also measured. There was no decrease in the degree of stimulation of the phospholipase by this nucleotide after 24 h of culture. It is concluded that the loss of vasopressin and angiotensin II responses in cultured liver cells is due in part to changes in receptors and also in their coupling to a guanine nucleotide binding protein.

Dissociation of tyrosine phosphorylation and activation of phosphoinositide phospholipase C induced by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 cells treated with platelet-derived growth factor.

Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) via phospholipase C-gamma1 (PLC-gamma1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC-gamma1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC-gamma1 translocation to the particulate fraction. Thus, although activation of PLC-gamma1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC-gamma1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC-gamma1 induced by PDGF and slowed the translocation of PLC-gamma1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP2 in cells labeled with myo[3H]inositol. The increased synthesis of PtdInsP2 could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC-gamma1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC-gamma1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC-gamma1, and on PtdInsP2 synthesis cannot be attributed to inhibition of PKC.

Purinergic agonist and G protein stimulation of phospholipase D in rat liver plasma membranes. Independence from phospholipase C activation.

Hormonal regulation of phospholipase D (PLD) was studied in isolated rat liver plasma membranes. Purinergic agents and a submaximal concentration of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), a non-hydrolyzable analog of GTP, synergistically stimulate phosphatidylethanol formation, a measure of PLD activity. The rank order of efficacy for stimulation of PLD activity in the presence of 0.2 microM GTP gamma S was beta, gamma-methylene-ATP > adenosine 5'-0-(3-thiotriphosphate) = ATP = ADP = 2-methylthio-ATP > alpha, beta-methylene-ATP = UTP. This pattern of activation does not conform to the series at known P2 receptors. GTP gamma S stimulated PLD activity in a dose-dependent manner, and the GTP gamma S dose-response curve for phosphatidylethanol formation was shifted to the left by an analog of ATP. Activation of PLD by purinergic agents in the presence of GTP gamma S supports the involvement of a purinergic receptor of the P2 class and a GTP-binding protein. Purinergic agents competitively inhibited [35S]adenosine 5'-0-(3-thiotriphosphate) binding to plasma membranes in the rank order adenosine 5'-0'(3-thiotriphosphate) > ATP > alpha,beta-methylene-ATP = UTP >> beta, gamma-methylene-ATP = ADP. Stimulation of phosphoinositide phospholipase C (PI-PLC) by purinergic agents, as measured by release of radioactivity from endogenously myo[3H]inositol-labeled plasma membranes, occurred in the order alpha, beta-methylene-ATP >> 2-methylthio-ATP. Beta, gamma-methylene-ATP had little effect on PI-PLC activity. Different dose-response relationships for agonist-stimulation of PI-PLC and PLD indicate that activation of PI-PLC is not involved in stimulation of PLD in rat liver plasma membranes, and suggest that purinergic activation of PLD occurs via a pathway involving a G protein and a heretofore uncharacterized P2 receptor.

Subcellular fractions of bovine brain degrade phosphatidylcholine by sequential deacylation of the sn-1 and sn-2 positions.

Phosphatidylcholine (PC) metabolism was investigated using cytosol (fraction I) and particulate fractions of bovine brain that were enriched with microsomes (fraction II), plasma membranes (fraction III) or mitochondria (fraction IV). Fractions I-III incubated with 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine yielded [14C]arachidonic acid at near equal rates, whereas only fraction I accumulated significant amounts of 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine. Much slower rates of arachidonic acid release were observed using an ether PC (1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine). Moreover, arachidonic acid yield from the diacyl, but not ether PC was slowed by pretreating fractions I-III, but not IV, with phenylmethylsulfonyl fluoride (PMSF). Coincident with this decreased arachidonic acid, 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine was increased, indicating high PLA1 activity. Taken together these data suggest that arachidonic release was largely dependent on initial deacylation of position sn-1. Incubating each untreated fraction with 2-[3-H]arachidonoyl-sn-glycero-3-phosphocholine yielded [3H]arachidonic acid (lysophospholipase A2 activity) at rate that was substantially greater than that using the comparable PMSF-treated fraction. Thus, the large effect of PMSF on arachidonic acid release can be accounted for if much of the fatty acid formation arose from the sequential sn-1 and sn-2 deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2. When PMSF-treated fractions were incubated with 2-[3H]arachidonoyl-sn-glycero-3-phosphocholine, [3H]PC accumulated at low rates that were enhanced by adding coenzyme A or stearoyl-coenzyme A. Thus, the lysophospholipid was also reacylated to form PC, but this reaction was negligible in the absence of PMSF and added cofactors. In summary, we conclude that, in brain subcellular fractions, deacylation of the sn-1 position of diacyl-PC proceeded more rapidly than sn-2 hydrolysis. There was substantial further metabolism of 2-acyl lysophospholipids due to the combined activities of a PMSF-sensitive and -insensitive lysophospholipase. Finally, the sequential deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2 probably accounted for the major portion of arachidonic acid produced.