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J H Exton

Publications and source records attributed to J H Exton.

At least 109 records · Page 6Linked to original sources
PubMed1991

Activation of phospholipase D: a signaling system set in motion by perturbation of the T lymphocyte antigen receptor/CD3 complex.

A number of cellular signaling systems are called into play by interaction of the T lymphocyte antigen receptor/CD3 complex with its cognate antigen. Well-described signaling systems include phosphoinositide turnover, tyrosine phosphorylation, protein kinase C activation, and increased cytosolic calcium. We have explored the possibility that another recently described signaling system, activation of phospholipase D, may be operative. Data presented here demonstrate that stimulation of Jurkat T cells with anti-CD3 antibodies or phorbol esters resulted in activation of phospholipase D, as measured by production of phosphatidylethanol and phosphatidic acid. The combination of anti-CD3 antibody plus phorbol ester led to a greater than additive production of phosphatidylethanol and to the additive production of phosphatidic acid (in the absence of ethanol). Phorbol esters as a second stimulus with anti-CD3 antibody led to a additive increase in cellular diacylglycerol content but provided no increased production of inositol phosphates, suggesting that diacylglycerol production in these cells results from hydrolysis of noninositol containing lipids as well as from phosphinositides. Exogenous addition of phosphatidic acid led to increases in cytosolic calcium that, depending on the concentration used, resulted from release of an intracellular store of calcium and influx of extracellular calcium. Changes in cytosolic calcium occurred in the absence of inositol phosphates production. These studies establish a role for increased phospholipase D activity in T lymphocyte activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Some thoughts on the mechanism of action of insulin.

Proposed mechanisms by which insulin exerts its effects are discussed. Evidence for a role for the tyrosine kinase activity of the insulin receptor and of a phosphorylation/dephosphorylation cascade is presented. The possible roles of phospholipid breakdown, diacylglycerol, and protein kinase C are discussed. The hypothesis that insulin elicits the hydrolysis of a glycosyl phosphatidylinositol to form a mediator of certain of its actions is considered in detail. The evidence that a G protein is involved in insulin action is analyzed.

Animals↗
PubMed1990

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.

Angiotensin II↗
PubMed1990

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.

Animals↗
PubMed1990

Purification from bovine liver membranes of a guanine nucleotide-dependent activator of phosphoinositide-specific phospholipase C. Immunologic identification as a novel G-protein alpha subunit.

Cholate-solubilized extracts from bovine liver plasma membranes preincubated with the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) displayed enhanced phosphoinositide-specific phospholipase C activity compared with extracts from membranes incubated without nucleotide or with ATP or GDP analog. Resolution of the GTP gamma S-elicited activator of phospholipase C was achieved using heparin-Sepharose which bound the phospholipase C activity. Recombination of non-adsorbed extract with salt-eluted phospholipase C activity resulted in a stimulation of enzyme activity. The GTP gamma S-dependent activator was purified, on the basis of its ability to activate partially purified phospholipase C, by sequential chromatography on Q-Sepharose, Sephacryl S-300, octyl-Sepharose, and Mono Q. The presence of G-protein beta subunits and the alpha subunits of Gi1, Gi2, and Gi3 was detected, by immunoblot analysis, in Mono Q-purified phospholipase C activator preparations. Resolution of the activator from these alpha subunits was achieved by incubation with pertussis toxin in the presence of millimolar NAD+ followed by rechromatography on Mono Q. The phospholipase C activator, thus resolved from ADP-ribosylated alpha i subunits, possessed an approximate Mr of 42 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copurified with a substoichiometric amount of beta subunit. Immunoblot analysis of fractions from the final Mono Q column revealed cross-reactivity of the 42-kDa phospholipase C activator with antipeptide antibodies raised against residues 160-169 of alpha i1 and a region of sequence common to all known G-protein alpha subunits. The 42-kDa activator was not recognized by other alpha subunit-specific or common antibodies. These findings identify the purified phospholipase C activator as a novel G-protein alpha subunit. This may represent the active subunit of the pertussis toxin-insensitive G-protein mediating receptor-stimulated phosphoinositide breakdown in mammalian liver.

Adenosine Diphosphate Ribose↗
PubMed1990

Signaling through phosphatidylcholine breakdown.

Several mechanisms of agonist control of PC hydrolysis have been described: control by G proteins, protein kinase C, Ca2+, and tyrosine kinases. The relative importance of these mechanisms remains to be demonstrated. Another major point is the physiological significance of PC hydrolysis. The prolonged formation of DAG from PC may be important in cellular control mechanisms that require long term activation of protein kinase C. The functions of PA are presently unknown, but the rapid formation of high concentrations of this lipid during stimulation by agonists strongly suggests that it has signaling functions. It is obvious that much work remains to define the physiological significance of agonist-stimulated PC breakdown.

Diglycerides↗
PubMed1989

Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown.

The molecular species of 1,2-diacylglycerol in control and agonist-stimulated rat hepatocytes were analyzed by high performance liquid chromatography. Twelve species were identified which were increased nonuniformly by 100 nM vasopressin. Most species were increased 2-3-fold, but some (C16:0/C20:4 and C18:0/C20:4) were increased 3-6-fold. Selectively greater increases in the latter two species were also induced by ATP, angiotensin II, and A23187 ionophore, however, phorbol ester caused uniform increases. Calcium depletion of the cells with chelator resulted in a uniform 2-fold effect of vasopressin on 1,2-diacylglycerol species, with greater increases in C16:0/C20:4 and C18:0/C20:4 being restored by Ca2+ readdition. Comparison of the increases in 1,2-diacylglycerol species caused by the Ca2+-mediated agents with the molecular species present in rat hepatocyte phospholipids supports the concept that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates. In hepatocytes incubated for 5 min to 2 h with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine, the label was incorporated mainly into phosphatidylcholine, and subsequent incubation with vasopressin, angiotensin II, ATP, epinephrine, A23187, and phorbol ester caused formation of [3H]alkyl-acylglycerol, but not [3H]alkyl-phosphatidic acid. The time course and concentration dependence of the vasopressin effect were similar to those reported previously for total 1,2-diacylglycerol (Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260, 14201-14207). Calcium depletion induced by chelator inhibited the effect of vasopressin, and readdition of Ca2+ largely restored the effect. In cells incubated with [14C]lyso-phosphatidylcholine, [3H]phosphatidylcholine, or [14C]phosphatidylethanolamine for 5 or 30 min to label hepatocyte phosphatidylcholine, vasopressin also induced the formation of labeled 1,2-diacylglycerol, but not phosphatidic acid. In contrast, in hepatocytes prepared from rats injected intraportally with [3H]alkyl-lyso-glycerophosphocholine 20 h previously, the hormone induced the rapid formation of both labeled 1,2-diacylglycerol and phosphatidic acid. In summary, these isotopic data indicate that a rapidly labeled pool of phosphatidylcholine is hydrolyzed to 1,2-diacylglycerol and a slowly labeled pool is broken down to both 1,2-diacylglycerol and phosphatidic acid in hepatocytes stimulated by Ca2+-mobilizing agents. It is concluded from both the analyses of molecular species of 1,2-diacylglycerol and the labeling experiments that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates in hepatocytes stimulated with Ca2+-mobilizing agonists and that the mechanisms responsible may involve both Ca2+ and protein kinase C.

Adenosine Diphosphate↗
PubMed1989

Changes in the concentration and fatty acid composition of phosphoinositides induced by hormones in hepatocytes.

The hormonal regulation of phosphoinositide levels in isolated hepatocytes was studied using chemical means. Extracted inositol phospholipids were adsorbed to neomycin-coated glass beads and then eluted and quantitated by charring after separation by thin layer chromatography on silica gel. The amounts (in nanograms/mg wet weight) of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) were 20 +/- 1, 16 +/- 1, and 1790 +/- 140, respectively). Incubation of the cells with 100 nM vasopressin decreased the value for PIP2 to 10 +/- 0.2 at 15 s, 12 +/- 1.5 at 1 min, and 14 +/- 2.1 at 5 and 30 min. In contrast, the hormone increased 1,2-diacylglycerol plus phosphatidate by over 200 ng/mg wet weight at 5 min under similar conditions (Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309-15315). PIP2 was also significantly decreased at 15 s by angiotensin II (100 nM), ATP (100 microM), and epinephrine (1 microM). In contrast, PIP was not significantly changed, and PI was significantly decreased (by approximately 15%) at later times (15 and 30 min). The changes in phosphoinositide mass were well correlated with changes in labeled phosphoinositides in hepatocytes previously incubated with [3H]inositol for 90 min. The amounts of inositol phospholipids in liver plasma membranes (in micrograms/mg protein) were 2.1 +/- 0.2 for PIP2, 0.24 +/- 0.03 for PIP, and 23 +/- 4 for PI. Comparison of these values with those for whole cells suggests that PIP2 is enriched in the plasma membrane, whereas PIP is present elsewhere in the cell. The fatty acid composition of whole cell PIP2 showed significant differences from that of PI. The percentages of palmitic, stearic, linoleic, and arachidonic acids were, respectively, 14, 41, 10, and 25 for PIP2 and 10, 34, 7, and 37 for PI. Vasopressin treatment for 15 s did not alter the fatty acid composition of PIP2. The corresponding fatty acid percentages for liver plasma membranes were 13, 41, 11, and 21 for PIP2 and 8, 34, 0, and 40 for PI. The fatty acid composition of PIP in whole cells and plasma membranes resembled that of PIP2.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1989

An enzymatic assay for picomole levels of phosphatidate.

An assay for phosphatidate that is sensitive, specific, and highly reproducible is described. Phosphatidate is transacylated with methylamine and the glycerol 3-phosphate produced is measured enzymatically. The assay is linear from 50 to 1500 pmol of phosphatidate and can be used to quantitate phosphatidate in small amounts of biological materials.

Animals↗
PubMed1989

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.

Adenosine Diphosphate Ribose↗
PubMed1989

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.

Adenosine Diphosphate Ribose↗
PubMed1988

Characterization of the angiotensin II receptor in primary cultures of rat hepatocytes. Evidence that a single population is coupled to two different responses.

The angiotensin II receptor of cultured rat hepatocytes was characterized using [3H]angiotensin II as radioligand. Binding at 23 degrees C was rapid (t1/2 = 0.65 min) with equilibrium being reached in 10-12 min. At this time, binding was completely reversible after 20 min (t1/2 = 3.5 min), indicating negligible internalization of the ligand. Analysis of the saturation binding curve showed one population of binding sites with an apparent KD of 8.6 nM and a Bmax of 35 fmol/mg of protein. The time courses of association and dissociation were also consistent with one class of binding sites with an apparent kinetically derived KD of 7.7 nM. The order of potency of different agonists and antagonists to increase cytosolic Ca2+ or phosphorylase a or inhibit the effects of angiotensin II on these parameters was the same as for their mimicry or reversal of angiotensin II inhibition of glucagon-induced cAMP accumulation, and was well correlated with their order of potency to inhibit angiotensin II specific binding. Treatment of cultured hepatocytes with dithiothreitol caused a time- and concentration-dependent inhibition of angiotensin II binding and corresponding alterations of angiotensin II effects on phosphorylase and cAMP. It also inhibited the actions of other hormones on phosphorylase. These results indicate that hepatocytes contain a homogeneous population of angiotensin II receptors that are coupled to two different biological effects apparently mediated by different G-proteins.

Angiotensin II↗
PubMed1988

Agonist-induced down-regulation of the angiotensin II receptor in primary cultures of rat hepatocytes.

The ability of angiotensin II to down-regulate its receptor was tested on rat hepatocytes in primary culture for 4 h. Angiotensin II treatment decreased [3H]angiotensin II specific binding in a concentration- and time-dependent manner. The effect was maximum with 1 microM angiotensin II and after 2 h. There was a decrease in the maximum number of binding sites (56% of control) with no significant effect on the apparent dissociation constant. The down-regulation was blocked by the angiotensin II antagonist [Val4,Ile7]angiotensin III and was not induced by other hormones (e.g. vasopressin, norepinephrine, or glucagon) or by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate or A23187 ionophore. The decrease in angiotensin II receptors resulted in correlated decreases in the potency of angiotensin II to activate phosphorylase or lower glucagon-induced cAMP accumulation. However, high concentrations of the agonist were still able to elicit maximal responses in both parameters. Down-regulation of the receptor was not dependent upon active Gi, since it was still observed after ADP-ribosylation and inactivation of Gi by pertussis toxin. The above results indicate that the down-regulation of the hepatic angiotensin II receptor induced by its agonist is homologous and does not involve Gi, Ca2+, or protein kinase C. The correlation of receptor loss with decreases in the potency of angiotensin to activate phosphorylase and inhibit glucagon-induced cAMP accumulation is consistent with the idea that a single receptor population regulates two different messengers, i.e. calcium and cAMP.

Angiotensin II↗
PubMed1988

The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists.

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.

Adenosine Triphosphate↗