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Biomedical subjects

S T Crooke

Publications and source records attributed to S T Crooke.

At least 127 records · Page 7Linked to original sources
PubMed1988

Inhibition of formation of cyclic AMP and cyclic GMP by vasopressin in smooth-muscle cells is insensitive to pertussis toxin.

Pretreatment of A-10 cells with pertussis toxin had no effect on [arginine]vasopressin-mediated inhibition of cyclic nucleotide accumulation. Pretreatment of the cells with the same concentration of pertussis toxin produced 90-95% inhibition of [32P]ADP ribosylation in membranes, suggesting that these cells possess pertussis-toxin substrate and that the toxin enters the cells to reach its site of action. The functional integrity of the pertussis-toxin substrate in these cells is confirmed by the observation that in these cell membranes increasing concentrations of GTP inhibited basal, forskolin- and NaF-stimulated adenylate cyclase activities, and this inhibition was abolished when the cells were pretreated with pertussis toxin. In addition, thrombin-mediated inhibition of isoprenaline-stimulated cyclic AMP accumulation was also inhibited by pertussis-toxin pretreatment of the cells. These data suggest that, unlike thrombin, [arginine]vasopressin-induced inhibitory effects on cyclic nucleotide accumulation in smooth-muscle cells are not mediated by pertussis-toxin substrate.

Adenosine Diphosphate Ribose↗
PubMed1988

Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.

Adenylyl Cyclases↗
PubMed1988

Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C.

We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.

Amino Acid Sequence↗
PubMed1988

Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes.

Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.

Adenylyl Cyclases↗
PubMed1988

Ubiquitin carboxyl-terminal peptides. Substrates for ubiquitin activating enzyme.

The carboxyl terminus of ubiquitin is activated in the presence of ATP to enter the ubiquitin cycle in cells. Peptides corresponding to the COOH-terminal region of ubiquitin were synthesized to investigate their effects on the ATP/ubiquitin-dependent proteolytic pathway. Their activities in the PPi exchange assay with ubiquitin activating enzyme (E1) were proportional to their length. The hexapeptide Ac-Leu-Arg-Leu-Arg-Gly-Gly reacted with ATP to form an enzyme-adenylate-hexapeptide complex and at high concentrations was 20-25% as active as human ubiquitin in the PPi exchange assay with E1. However, the hexapeptide was not transferred to the sulfhydryl "thiol" site on E1. In addition, the COOH-terminal peptides did not support the degradation of 125I-bovine serum albumin in the reticulocyte lysate system. A nonhomologous peptide of equivalent length was inactive in all assays. Thus, synthetic COOH-terminal peptide(s) of ubiquitin can partially substitute for ubiquitin in its reactions with E1 but do not support subsequent steps of the energy-dependent proteolytic pathway. These results show that it may be possible to design small molecules that either serve as substrates or inhibitors for other specific steps in ubiquitin-dependent pathways.

Adenosine Triphosphate↗
PubMed1988

Leukotriene D4-induced activation of protein kinase C in rat basophilic leukemia cells.

We studied the subcellular distribution of protein kinase C (PKC) in the particulate and cytosolic fractions of rat basophilic leukemia (RBL-1) cells treated with leukotriene D4 (LTD4) and compared these results with those of phorbol myristate acetate (PMA). Consistent with the earlier reports, treatment of RBL-1 cells with PMA resulted in a time- and dose-dependent translocation of PKC from cytosolic to the particulate fractions, sustained for at least 10 min. When RBL-1 cells were treated with LTD4, a small, transient decrease in PKC activity in cytosolic fraction was observed within 7.5 s after LTD4 treatment. This was accompanied by a significant increase of PKC in the particulate fraction. However, at 15 and 30 s, both particulate and cytosolic PKC activities were increased with LTD4 treatment. The activation induced by LTD4 was dose- and time-dependent with maximal effects occurring within 30 s, declining at the later time points. Pretreatment of the cells with 2(R)-hydroxy-3(S)-carboxyethylthio-3-[2-(8-phenyloctyl,pheny l]propanoic acid (SK&F 104353), a high affinity specific LTD4 receptor antagonist, and also with staurosporine, a potent inhibitor of PKC, completely inhibited the LTD4-induced activation of PKC both in the particulate and cytosolic fractions. These results suggest that activation of PKC by LTD4 is different from that elicited by PMA. The ability of SK&F 104353 to block LTD4-induced activation of PKC further suggests that stimulation of PKC might be an important intermediate step in the signal transduction mechanism of the LTD4 receptor in RBL-1 cells.

Animals↗
PubMed1988

Vasopressin induces V1 receptors to activate phosphatidylinositol- and phosphatidylcholine-specific phospholipase C and stimulates the release of arachidonic acid by at least two pathways in the smooth muscle cell line, A-10.

The rat thoracic aortic smooth muscle cell line, A-10, expresses vasopressin receptors of the V1 subtype. Vasopressin treatment of these cells stimulated the release of arachidonic acid and the formation of diacylglycerol and phosphocholine. These responses to vasopressin were inhibited by the V1-specific antagonist SK&F 100273, indicating that these were receptor-mediated phenomena. The mechanisms by which V1 receptors mediate arachidonic acid release appeared to be unaffected by cycloheximide or actinomycin D, suggesting that the release is independent of protein and RNA synthesis. The V1 receptors also appeared to be coupled to a phospholipase C which can hydrolyze phosphatidylcholine, a possible source of the released arachidonic acid. Phosphocholine and diacylglycerol were also generated. The release of arachidonic acid, phosphocholine, or diacylglycerol was not affected by prior treatment of the cells with pertussis toxin (islet-activating protein). Thus, the release of these second messengers is not mediated by the guanine nucleotide-binding protein Gi or other pertussis toxin-sensitive substrates. We conclude that V1 receptors induce the release of arachidonic acid and the formation of diacylglycerol and phosphocholine via the activation of both a phosphatidylinositol- and phosphatidylcholine-specific phospholipase C.

Animals↗
PubMed1988

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.

Animals↗
PubMed1988

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase.

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.

Adenylyl Cyclases↗
PubMed1988

Selective purification of cardiac myosin by a high-performance salicylate affinity column.

The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin.

Amino Acids↗
PubMed1988

Leukotriene D4-induced homologous desensitization of calcium mobilization in rat basophilic leukemia cells.

Previous work from our laboratory demonstrated that RBL-1 cells express membrane receptors for leukotriene (LT)D4 and that agonist stimulation of these receptors results in mobilization of intracellular calcium ([Ca++]i). We have used this LTD4 receptor-mediated event to examine the effects of homologous desensitization in RBL-1 cells. Prior exposure of RBL-1 cells to LTD4 resulted in a 40% reduction in the amount of maximal [Ca++]i mobilization. This desensitization was concentration- and time-dependent, with an EC50 of 1 to 3 nM and with 50% of the desensitization occurring after 7.5 min of pretreatment. SKF 104353 (100 nM), a high affinity, LTD4-receptor antagonist, blocked completely LTD4-induced desensitization of RBL-1 cells. The LTD4-induced desensitization was stereospecific, as the (5R,6S)-enantiomer of LTD4 was at least 100 times less potent than LTD4. Pretreatment of RBL-1 cells with LTD4 did not alter the ability of thrombin or ATP to induce [Ca++]i mobilization, suggesting that the desensitization was of the "homologous" type. The density of [3H]LTD4 receptors in RBL-1 cell membranes was decreased 23% by prior treatment of RBL-1 cells with LTD4. These results demonstrate that the [Ca++]i mobilization induced by LTD4 can be desensitized as a result of prior exposure to LTD4 and that the LTD4 receptor and/or second messenger systems can be specifically down-regulated.

Animals↗
PubMed1988

Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells.

A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. [3H]LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the [3H]myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of [3H]inositol-trisphosphate was rapid and the induction of biosynthesis of [3H]inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

Animals↗
PubMed1988

Staurosporine inhibits protein kinase C and prevents phorbol ester-mediated leukotriene D4 receptor desensitization in RBL-1 cells.

The aim of the present study was to investigate the effects of staurosporine on phorbol-myristate acetate (PMA)-induced activation of protein kinase C (PKC) and the desensitization of leukotriene D4 (LTD4)-stimulated Ca2+ mobilization in rat basophilic leukemia (RBL-1) cells. Staurosporine, one of the most potent PKC inhibitors known to date, markedly inhibited partially purified PKC from RBL-1 cells with an IC50 of 3 nM. Exposure of RBL-1 cells to PMA resulted in inhibition of LTD4-stimulated Ca2+ mobilization. However, prior treatment of the cells with staurosporine completely prevented PMA-induced desensitization of LTD4-stimulated Ca2+ mobilization. This reversal of Ca2+ desensitization by staurosporine was dose dependent with an IC50 of 0.1 microM. Treatment of RBL-1 cells with PMA resulted in translocation and activation of PKC from the cytosol to the membrane fraction. Pretreatment of RBL-1 cells with staurosporine inhibited the PMA-induced activation of PKC in the membrane fraction. The inhibition of PKC activity by staurosporine was time and dose dependent with an IC50 of 0.9 microM. These results show that PMA-induced heterologous desensitization is mediated by PKC and staurosporine prevented this process by directly inhibiting PKC in intact RBL-1 cells.

Alkaloids↗
PubMed1988

Leukotriene B4-induced homologous desensitization of calcium mobilization and phosphoinositide metabolism in U-937 cells.

Differentiated U-937 cells express leukotriene B4 (LTB4) receptors and mobilize Ca++ in response to LTB4. Using this cell line, we have characterized LTB4-induced desensitization. Prior exposure of U-937 cells to LTB4 resulted in a concentration- and time-dependent decrease in Ca++ mobilization in response to a subsequent challenge with LTB4 (EC50 = 2 nM; T1/2 = 4 min). Desensitization was temperature-dependent, occurring in cells pretreated with LTB4 at 37 degrees C but not at 4 degrees C. LTB4 pretreatment (100 nM, 30 min) decreased the maximal LTB4-induced Ca++ mobilization by 50% and increased the EC50 5-fold. After the cells were treated with LTB4, Ca++ mobilization in response to LTD4, platelet activating factor and chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were not decreased, suggesting that the desensitization was homologous and specific for LTB4. In U-937 cells, LTB4 and LTD4 induced concentration- and time-dependent and receptor-mediated phosphoinositide (PI) hydrolysis, which correlated with Ca++ mobilization. When U-937 cells were pretreated with LTB4, the amount of intracellular PI metabolites formed in response to LTB4 was reduced, whereas the response to LTD4 was unchanged. Examination of LTB4 membrane receptors in U-937 cells indicated that LTB4 pretreatment resulted in a 15% decrease in receptor number and a 3-fold decrease in affinity for LTB4. These results clearly demonstrate that LTB4-induced Ca++ mobilization and PI metabolism can be desensitized by prior exposure to LTB4 and that the mechanism of desensitization may involve altered affinity in agonist binding to the receptor.

Animals↗
PubMed1988

Leukotriene D4-induced homologous desensitization in basal and differentiated U-937 cells: characterization with the partial agonist leukotriene E4 and assessment of receptor reserve.

U-937 cells express receptors for leukotriene D4, and LTD4 stimulates receptor-mediated Ca++ mobilization with an EC50 of 5 nM. In these cells, LTE4 produces Ca++ mobilization with an EC50 of 500 nM and a maximum response that is 15 to 20% of the LTD4 response. LTE4 was shown to be a partial agonist, with a Kp of 400 nM and a linear relationship between effect and receptor occupancy, and was used as a tool to study LTD4-induced desensitization. Pretreatment of U-937 cells with LTD4 resulted in a concentration-dependent shift to the right and a decrease in the maximum of LTE4 concentration-response curves, indicating that desensitization had occurred. LTD4-induced desensitization was homologous for LTE4 and LTD4; Ca++ mobilization produced by LTB4, N-formyl-L-methionyl-L-leucyl-L-Phe and platelet-activating factor was not affected. LTD4-pretreatment produced a concentration-related desensitization of LTE4-induced Ca++ mobilization in both basal and dimethyl sulfoxide-differentiated U-937 cells. However, the maximal desensitization was greater in basal cells (66% +/- 4%) than in differentiated cells (33% +/- 7%). LTD4 pretreatment resulted in a greater percentage of reduction of Ca++ mobilization produced by LTE4 than that produced by LTD4, suggesting that a receptor reserve exists for LTD4. The extent of receptor reserve was assessed in basal and differentiated U-937 cells by comparing LTD4 concentration-response curves from control and desensitized cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1988

Subcellular localization of leukotriene D4 receptors in sheep tracheal smooth muscle.

The distribution of [3H]leukotriene D4 [( 3H]LTD4) receptors in subcellular membrane fractions obtained from sheep tracheal smooth muscle was studied. Using differential centrifugation and discontinuous sucrose density gradient centrifugation, the subcellular membranes were separated into six fractions. The [3H]LTD4 receptor distribution profile in these fractions correlated with markers for the plasma membrane (5'-nucleotidase and alkaline phosphodiesterase) and did not correlate with markers for the mitochondria (cytochrome c oxidase and succinate-dependent cytochrome c reductase). The dissociation constant (Kd) and maximum number of binding sites (Bmax) for [3H]LTD4 binding to the receptors in the crude mixture of membranes (PII) were 0.38 +/- 0.2 nM and 77 +/- 14 fmol/mg of protein, respectively. The Kd and Bmax of [3H]LTD4 binding to the receptors in the plasma membrane-enriched fraction (FII) were 0.40 +/- 0.2 nM and 268 +/- 46 fmol/mg of protein, respectively. The specificity profile of the [3H]LTD4 receptors in the plasma membrane-enriched fraction was equivalent to that observed in the crude membrane and correlated with the agonist myotonic activities in the smooth muscle contraction assay system. Furthermore, the binding of [3H]LTD4 to the plasma membrane receptors was modulated by guanine nucleotides in a manner analogous to that observed in crude membranes, suggesting that agonist interaction with the receptors was regulated by guanine nucleotide binding protein. These results suggest that, in sheep tracheal smooth muscle, the plasma membrane is the primary location of specific LTD4 receptors.

Animals↗