Biomedical subjects

S D Shukla

Publications and source records attributed to S D Shukla.

At least 19 recordsLinked to original sources

PubMed1992

Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.

Alkaloids↗

PubMed1992

Ethanol modulates epidermal growth factor-stimulated tyrosine kinase and phosphorylation of PLC-gamma 1.

A431 cells have an abundance of Epidermal Growth Factor (EGF) receptors which possess intrinsic tyrosine kinase activity. Treatment of membranes isolated from A431 cells with EGF caused a 2-3 fold increase in phosphorylation of a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) which is a substrate for tyrosine kinase. Treatment of these membranes with 0.1 to 100 mM ethanol altered basal tyrosine kinase activity in a biphasic manner; increase at 10 mM and decrease at 100 mM ethanol. The treatment of the membranes with the same concentrations of ethanol also altered EGF's ability to stimulate tyrosine kinase activity: increase at 0.1 mM ethanol and decrease at 10 mM. Strikingly, EGF-stimulated tyrosine kinase was more sensitive to ethanol than the basal activity. Experiments with other alcohols showed a relationship between chain length and the inhibitory ability of the alcohol. These data demonstrate a biochemical effect of low concentrations of ethanol on tyrosine kinase. Interestingly, ethanol treatment of A431 cells inhibited EGF-stimulated phosphorylation of PLC-gamma 1 which is a substrate for EGF receptor tyrosine kinase. It is concluded that ethanol at low concentrations has significant modulatory effect on basal and EGF-stimulated tyrosine kinase, as well as PLC-gamma 1 phosphorylation.

Amino Acid Sequence↗

PubMed1992

Hypersensitivity of diabetic human platelets to platelet activating factor.

Platelet activating factor (PAF) stimulated aggregation and [32P]-phosphatidic acid (PA) production was compared in normal and diabetic human subjects in platelet rich plasma. The concentration of PAF for half maximal (50%) aggregation of normal and diabetic platelets was 50 nM and 8 nM, respectively. PAF stimulated [32P]-PA production (a metabolite of phospholipase C pathway) was also greater in the platelets from diabetic subjects. This [32P]-PA production was inhibited by the PAF receptor antagonists SRI-63441 and SRI-63675. When the levels of glycosylated hemoglobin (HbA1c) were compared with the PAF stimulated [32P]-PA production a significant relationship was observed. These studies have demonstrated for the first time that diabetic human platelets show hypersensitivity to PAF in both aggregation and [32P]-PA production compared to normal subjects. This may be a result of some modification in phospholipid turnover mechanism and is receptor mediated. Further, the relationship of the degree of aggregation and [32P]-PA production to the level of HbA1c suggest that the insulin deficiency may contribute to these effects.

Blood Platelets↗

PubMed1992

Platelet-activating factor receptor and signal transduction mechanisms.

Platelet-activating factor (PAF) is the most potent phospholipid agonist known to date. Radioligand binding studies using [3H]PAF and structurally different PAF antagonists have provided the characteristics of PAF receptor(s) and its heterogeneity. Although efforts have been made to isolate the receptor, it was not until the recent cloning of the PAF receptor that the molecular architecture of the receptor can be visualized. The receptor shows homology to the G protein-coupled receptors with seven transmembrane spanning segments. Several serine, threonine, and tyrosine residues are present at the cytoplasmic side, which could serve as sites for phosphorylation. PAF activates GTPase, causes phospholipid turnover via phospholipases C, D, and A2 pathways and also activates protein kinase C and tyrosine kinase. Further, PAF stimulates Ca2+ mobilization some of which may occur via receptor operated channel. Second messengers generated by these multiple signalling pathways play role (or roles) in PAF responses and in the PAF induced expression of primary response genes. These recent developments throw light on the PAF receptor and its signal transduction mechanisms.

Animals↗

PubMed1991

Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane.

We have investigated the characteristics of platelet-activating factor (PAF)-stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets.

Animals↗

PubMed1991

Release of a membrane surface glycoprotein from human platelets by phosphatidylinositol specific phospholipase(s) C.

Phosphatidylinositol (PI) specific phospholipase C (PIase C) treatment of human platelets caused release of a surface glycoprotein in the medium. Human blood platelets were isolated by low speed centrifugation and surface glycoproteins were labelled with periodate/[3H]borohydride procedure. Intact surface-labelled platelets were treated with PIase C purified from culture filtrates of Staphylococcus aureus (SA) or Bacillus thuringiensis (BT). After PIase C treatments platelets were spun at low speed, pellet and supernatant were separated. The supernatant was further centrifuged at high speed (140,000 x g) for 30 min. The resulting supernatant and the pellet from low speed were subjected to SDS-PAGE analysis. Protein patterns were obtained by fluorography. Release of a specific glycoprotein of approx. 150 kDa in the medium was observed due to the PIase C treatment. Prolonged incubation of platelets in 0.25 M sucrose and depletion of NaCl concentrations also affected the release of this glycoprotein. BT-PIase C released more approx. 150 kDa protein than SA-PIase C. Western blot experiment with a monoclonal antibody (mAB), epitope SZ2, reactive to human platelet surface glycoprotein Ib (GPIb) complex, confirmed that released 150 kDa glycoprotein reacted with mAB of GPIb. The release of this protein by PIase C was not inhibited by proteinase inhibitors (EDTA, PMSF and leupeptin). Treatment of human platelet membranes with PIase C also caused release of this glycoprotein as evidenced by reactivity to GPIb-mAB. These studies demonstrate that PIase C treatment causes release of 150 kDa glycoprotein from human platelet membrane surface. It is suggested that 150 kDa glycoprotein is anchored to PI in human platelets and that this glycoprotein represents the GPIb complex.

Antibodies, Monoclonal↗

PubMed1991

Altered phospholipase activities related to alpha 1-adrenergic receptor supersensitivity of aortas from aldosterone-salt hypertensive rats.

Many of the concepts presented in this paper are summarized in Fig. 7. Some aspects are well supported while others are speculative. The operation of PLC in VSM is well established, and in some hypertensive models (AHR, SHRSP) PLC assays exhibited altered activation. Currently this pathway leading to the production of IP3 and DAG is considered to be the major regulator of Ca release from sarcoplasmic reticulum (SR) and Ca entry by channels (CaC). Regulation of PKC by [Ca]i and DAG is thought to play a major role in controlling Ca entry. PKC has also been proposed to regulate PLA2 as well as PLD in conjunction with elevated [Ca]i. An important issue to be resolved is whether receptor regulation of other lipases occurs independently of the PLC-[Ca]i-PKC axis. Currently information supporting receptor regulation is lacking for VSM, but few studies have been conducted. Our observation that NE stimulation of PLD activity occurs in VSM indicates that the control of VSM by biochemical messengers is much more complicated than previously proposed. This seemingly redundant pathway may allow VSM to use alternate substrates for producing PA and DAG than are readily available to PLC. It also allows PA to be produced directly without phosphorylation of DAG. Although the role of PA in the regulation of Ca entry was proposed earlier, definitive studies establishing this linkage are still required. Any PLD activity on PIP2 would produce biochemical messengers (PA, DAG) which could stimulate Ca entry without producing the messenger, IP3, associated with Ca release (inactive IP2 would be produced). If PLC and PLD were independently regulated by receptor-guanine nucleotide-regulatory protein (G-protein) complexes, this would offer the potential for some agonists to excite VSM by Ca release and Ca entry mechanisms while others may excite by Ca entry alone. This system would also circumvent the problem of limited substrate for cellular regulation of [Ca]i if PIP2 were the primary substrate. This limitation does not exist with other phospholipids such as phosphatidylcholine which is a preferred substrate for PLD. The presence of multiple phospholipases under separate receptor regulation allows for a wider range of tissue responses to various agonists, than a system which is linked only through the PLC-[Ca]i-PKC axis. The presence of a PLD pathway also reopens the interpretation of previous studies which demonstrated a resetting between receptor occupancy and production of second messengers by PLC.(ABSTRACT TRUNCATED AT 400 WORDS)

Aldosterone↗

PubMed1991

Inositol phospholipid turnover in PAF transmembrane signalling.

In a variety of cells and tissues, platelet activating factor (PAF) stimulates phospholipase C catalyzed breakdown of phosphoinositides. This results in the generation of the second messengers, inositol trisphosphate and diglyceride. This process occurs independently of extracellular Ca2+. A number of PAF structural analogues, receptor antagonists and drugs have been utilized to pharmacologically probe the activation of phospholipase C. PAF stimulation of the phosphoinositide turnover was shown to be sensitive to pertussis toxin in some systems, but not in others. The involvement of guanine nucleotide binding protein(s) and tyrosine kinase(s) in this process have also been postulated. These developments give new insights into PAF-receptor function at the molecular level, and also provide leads towards a better understanding of the cellular responses to PAF.

Animals↗

PubMed1991

Phospholipase D in cell signalling and its relationship to phospholipase C.

Phospholipases C and D are phosphodiesterases which act on phospholipid head groups. Although the presence of these enzymes in living organisms has long been known, it is only recently that their role in cell signal transduction has been appreciated. The new developments on phospholipases D (PLD) are especially noteworthy, since these enzymes catalyze a novel pathway for second messenger generation. In a variety of mammalian cell systems, several biological or chemical agents have recently been shown to stimulate PLD activity. Depending on the system, activation of PLD has been suggested to be either dependent on, or independent of, Ca2+ and protein kinase C. PLD primarily hydrolyses phosphatidylcholine (PC) but phosphatidylinositol and phosphatidylethanolamine have also been reported as substrates. Different forms of endogenous PLD may also exist in cells. Exogenous addition of PLD causes alterations in cellular functions. In many instances, Ca2+ mobilizing agonists may stimulate both PLC and PLD pathways. Interestingly, several metabolites of these two enzymes are second messengers and are common to both pathways (e.g. phosphatidic acid, diglyceride). This has raised the issue of the interrelationship between these pathways. The regulation of either PLC or PLD by cellular components, e.g. guanine nucleotide binding proteins or protein kinases, is under intense investigation. These recent advances are providing novel information on the significance of phospholipase C and D mediated phospholipid turnover in cellular signalling. This review highlights some of these new discoveries and emerging issues, as well as challenges for future research on phospholipases.

Animals↗

PubMed1991

Platelet activating factor induces expression of early response genes c-fos and TIS-1 in human epidermoid carcinoma A-431 cells.

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.

Cycloheximide↗

PubMed1990

Bioconversion of leukotriene D4 by lung dipeptidase.

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.

Animals↗

PubMed1990

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: studies with genistein and monoclonal antibody to phosphotyrosine.

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation. The inhibition of PLC by Genistein raised the possibility of involvement of tyrosine kinase in PAF receptor-coupled PLC activation. Western blot analysis using monoclonal antibody to phosphotyrosine demonstrated that PAF stimulated the tyrosine phosphorylation of two major proteins of 50,000 and 60,000 molecular weight. When platelets were challenged with PAF after treatment with either Genistein or CV-6209 (a PAF receptor antagonist), the reactivity of these proteins to monoclonal antibody was inhibited. Phosphoamino acid analysis of Mr 50,000 and 60,000 proteins confirmed that PAF increased the phosphorylation of tyrosine residues in both Mr 50,000 and 60,000 proteins and that this was inhibited by Genistein. Thus, PAF caused a receptor-dependent phosphorylation of tyrosine residues on Mr 50,000 and 60,000 proteins. Based on these observations, it is concluded that tyrosine kinase is involved in the PAF receptor-coupled PLC activation and signal transduction mechanism.

Animals↗

PubMed1990

Characterization of a 66-kilodalton surface glycoprotein of the human corneal endothelium.

The pellet recovered after centrifugation (5000 X g) of human corneal endothelial homogenates was used as the source of membranes in these studies. A 66-kilodalton (kD) protein was identified as the most abundant protein in the particulate pellet by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The de novo synthesis of the 66-kD protein by endothelial cells was observed during culturing of human corneas in the presence of 35S-methionine. The 66-kD protein was found to be a plasma membrane protein based on several of its properties, ie, its solubility in CHCl3:CH3OH, its labeling as surface glycoprotein, and during exposure to a photoaffinity hydrophobic probe: 1-azido-4-125I-iodobenzene. Furthermore this protein could be released from the particulate pellet after treatment with phosphatidylinositol-specific phospholipase C, suggesting its anchorage via a phosphatidylinositol glycan linkage in the plasma membrane. Such anchorage of this protein was further confirmed by its labeling during culture of corneas in the presence of 3H-myoinositol. The glycoprotein nature of the 66-kD protein was evident from its labeling during surface glycoprotein labeling of endothelial cells, staining with periodic acid-Schiff stain, and binding to peanut agglutinin (PNA), and lotus agglutinin (LTA) on SDS-acrylamide gels. The 66-kD protein of endothelial particulate pellets recovered from corneas of donors of different ages showed an age-related increase in binding to PNA and LTA. This suggested an increased glycosylation of the 66-kD protein with aging. A polyclonal anti-66-kD protein antibody was used as a probe to determine the presence of this protein in the rabbit and bovine corneal endothelia by the Western-blot analysis. The 66-kD protein was detected in both rabbit and bovine endothelia, but an additional immunoreactive species of 17 kD was also observed which may be a processed product of the 66-kD protein.

Aging↗

PubMed1989

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor.

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF (100 nM for 5 seconds) stimulated incorporation of 32P into proteins and caused [3H]InsP3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [3H]InsP3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [3H]InsP3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF.

Alkaloids↗

PubMed1989

Desensitization of platelet-activating factor-stimulated protein phosphorylation in platelets.

Treatment of 32P-labeled rabbit platelets with platelet-activating factor (PAF) caused a time- and dose-dependent phosphorylation of several proteins including five major phosphorylated proteins with apparent molecular weights of 20,000, 35,000, 40,000, 65,000, and 150,000. Both PAF and thrombin caused a rapid increase followed by a decrease in phosphorylation of proteins, indicating the occurrence of a phosphorylation-dephosphorylation process. Four separate PAF receptor antagonists, CV-3988, CV-6209, SRI-63-441, and SRI-63-675 drastically reduced the PAF-stimulated protein phosphorylation. The order of potency was SRI-63675 greater than SRI-63441 greater than or equal to CV-6209 greater than CV-3988. These antagonists had no effect on thrombin-stimulated protein phosphorylation. Pretreatment of platelets with PAF (0.1 nM) completely abolished any further protein phosphorylation by the same concentration of PAF. PAF pretreatment shifted the dose response of protein phosphorylation by about 2 log units, to the right. When platelets were treated with PAF (10 nM) for 10 min, this abolished phosphorylation of proteins by any concentration of PAF. These studies indicated a homologous desensitization of protein phosphorylation. Interestingly, PAF-pretreated platelets still exhibited phosphorylation of proteins by thrombin. On the other hand, a lack of protein phosphorylation by PAF or thrombin was observed in platelets preexposed to thrombin and this demonstrated a heterologous desensitization. It is concluded that phosphorylation of proteins by PAF is a PAF receptor-coupled event and that this process is desensitized in platelets preexposed to PAF. The fact that both the activation of phosphoinositide-specific phospholipase C and the phosphorylation of proteins are desensitized in PAF-pretreated platelets suggests that a close "regulatory" intercommunication between these processes exists.

Animals↗

PubMed1989

Antagonism of platelet activating factor receptor binding and stimulated phosphoinositide-specific phospholipase C in rabbit platelets.

The objective of this study was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of phosphoinositide-specific phospholipase C (PLC) in rabbit platelets. Saturation binding curves for [3H]PAF indicated that the PAF receptor has a dissociation constant (KD) of 28.72 nM. In comparison, PAF-stimulated PLC activity, as monitored by [3H]inositol triphosphate production, increased at lower concentrations and had an half-maximal effective concentration (EC50) value of 1.5 nM. Unlabeled PAF inhibited [3H]PAF binding competitively and demonstrated two binding sites, a high affinity site with an inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 microM. The inhibitory effects of four PAF antagonists, CV-3988, CV-6209, SRI 63-441 and SRI 63-675 on the binding of [3H]PAF were compared to the effects of the antagonists on PAF-stimulated PLC activity. The four antagonists inhibited [3H]PAF binding almost completely whereas their ability to inhibit PAF-stimulated PLC activity varied. CV-3988, SRI 63-441 and SRI 63-675 had half-maximal inhibitory concentration (IC50) values of 0.28, 0.78 and 0.42 microM, respectively, whereas CV-6209 was more potent at inhibiting [3H]PAF binding (IC50 = 7.73 nM). The SRI 63-441 and SRI 63-675 inhibited PLC totally with an IC50 value of 0.78 and 1.27 microM, respectively. The CV-3988 and CV-6209 showed a maximal PLC inhibition of about 45% with "apparent IC50" values of 1.05 and 0.17 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Altered biochemical and functional responses in aorta from hypertensive rats.

Factors that lead to supersensitivity of vascular smooth muscle to norepinephrine during aldosterone-salt-induced hypertension in rats appear to reside beyond ligand-alpha-adrenergic receptor binding, which we have shown previously to be normal. The objective of this study was to determine whether significant shifts occur in the coupling between receptors and the production of putative second messengers. Measures of [3H]myo-inositol phosphates in aorta (endothelium removed) exhibited a concentration-dependent increase to norepinephrine, with the 50% response shifted significantly to the left in the hypertensive group (7.0 +/- 0.9 X 10(-7) M in 8 control rats vs 1.1 +/- 0.2 X 10(-7) M in 8 hypertensive rats; p less than 0.001). The production of [32P]phosphatidic acid was also shifted (6.5 +/- 2.5 X 10(-7) M in 16 control vs 1.9 +/- 0.8 X 10(-7) M in 12 hypertensive rats; p less than 0.05). The functional responses of 42K efflux and contraction to norepinephrine were also significantly shifted threefold to 15-fold in the hypertensive group (p less than 0.001), but the 50% response typically occurred at a 10 to 100 times lower concentration than that for the production of myo-inositol phosphates and phosphatidic acid. The amplification between receptor occupancy and functional responses apparently occurs beyond the production of phosphoinositide metabolites. The fivefold shift in the 50% response of biochemical end points for the hypertensive group accounted for most of the shift (sixfold) in the functional end points.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Desensitization of receptor-coupled activation of phosphoinositide-specific phospholipase C in platelets: evidence for distinct mechanisms for platelet-activating factor and thrombin.

Platelet-activating factor (PAF) receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) was studied in platelets that were made refractory, by short-term pretreatments, to either PAF or thrombin. Generation of [3H]inositol triphosphate ( [3H]IP3) was monitored specifically for this purpose. [3H]Inositol-labeled rabbit platelets that were incubated (10 min) with increasing concentrations of PAF and subsequently challenged by the same concentration of PAF had greatly diminished PLC activity ( [3H]IP3 production) as compared to controls. Platelets incubated (10 min) with fixed concentrations of PAF and then challenged with increasing concentrations of PAF had log-dose response curves of [3H]IP3 production progressively shifted to the right (i.e., to higher concentrations) and were depressed as the PAF pretreatment with 10 nM PAF became completely refractory to further PAF stimulation of PLC. Washing the pretreated platelets with either buffer or buffer containing 0.5% bovine serum albumin did not restore the PAF for 10 min), platelets remained fully responsive to thrombin (2 units/ml)-stimulated production of [3H]IP3. Platelets pretreated with increasing concentrations of thrombin (0.15-2 units/ml) for different times (5-40 min) became refractory to both thrombin and PAF. It is concluded that PAF receptor-coupled activation of PLC becomes refractory (desensitized) in platelets preexposed to PAF, whereas platelets pretreated with thrombin are desensitized to both thrombin and PAF. It is proposed that thrombin has two transmembrane pathways leading to the activation of PLC, one shared with PAF and another utilizing separate mechanistic inputs.

Blood Platelets↗