ABSTRACT
We have developed a system to permeabilize human umbilical vein endothelial cells in monolayer culture by application of a high-voltage electric field. The permeabilized preparation allows access of small molecules (M(r) < 1000) without loss of large cytosolic proteins. Electropermeabilized cells exocytose highly multimeric von Willebrand factor from secretory granules in response to added Ca2+ (EC50 = 0.8 +/- 0.02 microM), with levels comparable with those observed on stimulation of intact endothelial cells by physiological agonists. MgATP2- potentiates Ca(2+)-driven von Willebrand factor secretion. Other nucleoside triphosphates, but not non-hydrolysable analogues, can replace ATP. Electropermeabilized cells also synthesize and release prostacyclin in response to added Ca2+ (EC50 = 0.3 +/- 0.08 microM), but nucleoside triphosphates markedly inhibit, whereas nonhydrolysable GTP analogues increase, Ca(2+)-driven prostacyclin synthesis. We conclude that elevation of the intracellular [Ca2+] is sufficient to cause efficient exocytosis of von Willebrand factor from permeabilized cells, despite evidence that additional second messengers are needed in intact cells. We find no evidence in endothelial cells for a guanine nucleotide-binding protein promoting exocytosis, although one is clearly involved in stimulating Ca(2+)-driven prostacyclin synthesis.
Subject(s)
Endothelium, Vascular/metabolism , Epoprostenol/biosynthesis , Exocytosis , von Willebrand Factor/metabolism , Calcium/pharmacology , Cells, Cultured , Electroporation , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , HumansABSTRACT
Human blood platelets carry a high affinity, but low capacity, saturable system for the uptake of noradrenalhe. The uptake is partially Na(+) dependent but cannot be categorised as uptake. It is distinct from the uptake system responsible for 5-hydroxytryptamine transport into the platelet since the selective inhibitors of the platelet uptake system for 5-hydroxytryptamine (citalopram, paroxetine) Wer from those for the uptake system for noradrenaline (normetanephrine, methylisoprenaline). 5-hydroxytryptamine inhibits noradrenaline uptake but with properties inconsistent with competition for the same uptake system while noradrenaline does not inhibit 5-hydroxytryptamine uptake. Neither noradrenaline nor 5-hydroxytryptamine uptake by human platelets is inhibited by dopamine.
ABSTRACT
We have examined the action of a range of transition metal nitrosyl compounds in the inhibition of ADP-induced platelet aggregation. Inhibition results from the formation of the activated nitric oxide (NO) complex of guanylate cyclase, hence increasing platelet [cGMP]. Nitrosylation of guanylate cyclase may occur by release of NO from a nitrosyl complex, or, indirectly, by nitrosation of a thiol group followed by decomposition of the S-nitrosyl thiol to give NO. The latter process might be expected to be more efficient for compounds with a greater NO(+)character, and hence nitrosating ability, of the nitrosyl complex, but the results did not show a consistent relationship between NO character and the inhibitory potency on platelets. Inhibition of aggregation by Rousin's black salt, Na[Fe(4)S(3)(NO)(7)], was abolished by haemoglobin, and enhanced in the presence of M&B22948. These findings indicate that activation of guanylate cyclase is mediated by extracellular release of NO. For sodium nitroprusside, inhibition of platelet aggregation became progressively less sensitive to addition of haemoglobin, indicating that another process, such as release of cyanide, became significant as the incubation time was increased.
ABSTRACT
This study examined the rheological properties of fibrin gels formed by adding thrombin to plasma samples from 99 subjects with fibrinogen concentrations ranging from 1.45 to 4.14 g/l. A highly significant (r = 0.757; P < 0.001) inverse correlation was observed between plasma fibrinogen concentration and the extent of clot deformability as estimated from the final value of the storage modulus (G') of the fibrin gel when obtained by rheological analysis. A similarly significant correlation (r = 0.844; P < 0.001) was obtained using samples from 47 subjects in which fibrin cross-linking was blocked by addition of 0.1 mM iodoacetamide to inactivate factor XIIIa. The characteristics of the relationship between G' and fibrinogen concentration in the plasma samples was comparable with that observed when the fibrin gel was formed by adding thrombin to purified fibrinogen. These results suggest that the increased risk of myocardial infarction associated with an elevated plasma fibrinogen concentration may, in part, be explained on the basis of a decreased deformability of the fibrin clot formed.
Subject(s)
Blood Coagulation , Fibrinogen/metabolism , Myocardial Infarction/blood , Aged , Humans , Hydrogen-Ion Concentration , Iodoacetamide/pharmacology , Kinetics , Male , Middle Aged , Myocardial Infarction/epidemiology , Sodium Chloride/pharmacologyABSTRACT
White and brown rat adipocytes have been permeabilised by repeated exposure of the cells in suspension to high voltage electrical discharges. The resulting preparations were permeable to low molecular weight materials, e.g., cyclic AMP, propidium iodide, and were stable in suspension with little evidence of rapid resealing, or of gross damage to the cell membrane. Leakage of lactate dehydrogenase was not markedly enhanced except at voltages in excess of 2 kV cm-1 for brown adipocytes. Exogenously-added cyclic AMP stimulated lipolysis (measured as glycerol release) in the electropermeabilised adipocytes far more effectively than in intact adipocytes. In brown, but not in white, adipocytes this effect was enhanced by addition of millimolar ATP. The EC50 for stimulation of glycerol release by cyclic AMP was 0.2 microM in electropermeabilised brown adipocytes, and 2 microM and 40 microM in electropermeabilised white adipocytes obtained from weanling and adult rats respectively. The effect of cyclic AMP on lipolysis was enhanced by addition of an inhibitor of cyclic AMP phosphodiesterases and was reduced by addition of 5'-AMP, adenosine or inosine (in brown adipocytes). Addition of adenosine deaminase caused a small, but significant, enhancement of cyclic AMP-driven lipolysis. Catecholamine-driven lipolysis was observed in electropermeabilised brown and white adipocytes, especially in the presence of GTP. Adrenaline-, and to a lesser extent cyclic AMP-, driven lipolysis in electropermeabilised white adipocytes was inhibited by insulin. This effect of insulin was not enhanced by addition of GTP or of a metabolically stable GTP analogue. The results obtained establish the electropermeabilised preparation as suitable for analysis of signal transduction pathways in white and brown adipocytes.
Subject(s)
Adipose Tissue/drug effects , Epinephrine/pharmacology , Lipolysis/drug effects , Signal Transduction , Adipose Tissue/cytology , Adipose Tissue, Brown/drug effects , Animals , Cyclic AMP/pharmacology , Electric Stimulation , Female , Glycerol/analysis , Insulin/pharmacology , Male , Norepinephrine/pharmacology , Rats , Rats, Sprague-DawleySubject(s)
Blood Platelets/physiology , Exocytosis , Membrane Fusion , Blood Platelets/drug effects , Calcium/blood , Calcium/pharmacology , Cell Membrane/physiology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Electric Stimulation/instrumentation , Electric Stimulation/methods , Humans , Indicators and Reagents , Magnesium/blood , Rubidium/blood , Thrombin/pharmacologySubject(s)
Blood Platelets/physiology , Calcium/physiology , Adenosine Triphosphate/physiology , Cyclic AMP/physiology , Cyclic GMP/physiology , Cytosol/metabolism , Diglycerides/physiology , Epinephrine/physiology , GTP-Binding Proteins/physiology , Humans , Platelet Activation , Signal TransductionABSTRACT
Studies were performed to determine whether formation of platelet aggregates itself, could cause an increase in cytosolic [Ca(2+)]([Ca(2+)](i)) which is independent of that resulting from the addition of agonists which induce aggregation. An increase in [Ca(2+)](i) did not coincide with aggregate formation when this response was dissociated from the addition of ADP or thrombin by delay either in initiating stirring or, for ADP, in adding fibrinogen. No increase in [Ca(2+)](i) occurred when aggregation was induced by addition of 1,2-dioctanoylglycerol or of ristocetin, or for chymotrypsin-treated platelets by addition of fibrinogen. The results demonstrate clearly that aggregate formation does not cause an increase in [Ca(2+)](i), and therefore exclude this possibility as an explanation for the discrepancies observed when [Ca(2+)](i) is measured, using aequorin and Fura2 as probes and as an underlying mechanism to account for contact-induced responses.
ABSTRACT
In aspirin-treated platelets labelled by preincubation with [(3)H]-choline, enhanced release of both [(3)H]-choline and [(3)H]-choline phosphate resulted from stimulation by collagen or thrombin. No such release accompanied stimulation by ADP, platelet-activating factor or adrenaline. Release of [(3)H]-choline phosphate was entirely dependent on aggregate formation whereas release of [(3)H]-choline was reduced but not eliminated, if aggregation was prevented. The properties of [(3)H]-choline and [(3)H]-choline phosphate release indicated that both collagen and thrombin induced activation of phospholipase D in the absence of aggregate formation. Such activation was augmented if aggregate formation occurred. Aggregation induced by these two agonists also caused activation of phosphatidylcholine-specific phospholipase C. These effects were prevented in the presence of staurosporine and could also be induced by addition of a synthetic 1,2-diacylglycerol indicating a role for protein kinase C.
ABSTRACT
Increases in [cyclic-3',5'-GMP] in aspirin-treated platelet-rich plasma and washed platelet preparations resulted from stimulation by all excitatory agonists tested, and by other agents which induced aggregate formation. The maximal increase observed was approximately 4-fold above the resting level. The increase in [cyclic-3',5'-GMP] correlated closely in both time-, and agonist dose-dependence with aggregation as measured by an increase in light transmittance. It was delayed in time, and occurred at a higher agonist concentration, than the initial phase of aggregation as measured by loss of single platelets. The extent of increase in [cyclic-3',5'-GMP] was independent of the signal transduction pathway used by the agonist/agent. Inhibition of aggregation by removal of Ca(2+), failure to induce contact, addition of antibodies or antagonists to the glycoprotein IIb/IIIa complex or the presence of an inhibitory agonist such as PGI(2) prevented the increase in [cyclic-3',5'-GMP]. Contact with collagen fibrils causing adhesion to this matrix, or aggregate formation induced by ristocetin or by certain lectins also caused an increase in [cyclic-3',5'-GMP]. Contact of platelets either with other platelets or with a matrix therefore results in stimulation of guanylate cyclase. The mechanism responsible for such stimulation remains unclear but does not appear simply to be attributable to activation of nitric oxide synthase by Ca(2+).
ABSTRACT
When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 µM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).
ABSTRACT
The intracellular distribution and maximal activities of nine enzymes involved in the biosynthesis and degradation of citric acid in Aspergillus niger were determined under conditions of growth and of citric acid production. Under these conditions the intracellular location of the enzymes in most cases resembled that described for other filamentous fungi. Pyruvate carboxylase was found predominantly or exclusively in the cytosol. A single isoenzyme of NADP-isocitrate dehydrogenase was present, which appeared to be localised in the mitochondrion. No significant differences in maximal enzyme activities were observed except for NADP-isocitrate dehydrogenase, which showed decreased activity in production-phase mycelia. The results obtained support the scheme proposed by C.P. Kubicek for the intracellular organisation of citric acid formation but provide little evidence that this process is controlled at the level of the biosynthesis of any of the enzymes examined here.
Subject(s)
Aspergillus niger/enzymology , Citrates/metabolism , Aspergillus niger/growth & development , Kinetics , Pyruvate Carboxylase/metabolismABSTRACT
Adenylate kinase isoenzymes localised in the mitochondria and in the cytosol have been detected in extracts of glucose-grown Aspergillus nidulans using specific staining after electrophoresis on cellulose acetate. The isoenzymes have similar Km values for AMP, ADP and MgATP2- but may differ in the mechanism used for internucleotide phosphate transfer.
Subject(s)
Adenylate Kinase/analysis , Aspergillus nidulans/enzymology , Isoenzymes/analysis , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Adenylate Kinase/metabolism , Cytosol/enzymology , Isoenzymes/metabolism , Kinetics , Mitochondria/enzymologyABSTRACT
Addition of micromolar Ca2+ to electropermeabilized human platelets which had been pre-labelled with [3H]arachidonate causes release of 3H only when millimolar concentrations of a nucleoside triphosphate, e.g. ATP, are present in the incubation medium. Addition of millimolar Ca2+ in the absence of ATP, or preincubation with ATP before addition of micromolar Ca2+, fails to cause a significant increase in 3H release. Purine nucleotides are more effective than pyrimidine nucleotides in activating Ca2(+)-driven 3H release. This activation does not appear to involve phosphate transfer, since metabolically stable analogues of ATP, e.g. the beta gamma-imido analogue, are effective in promoting 3H release.
Subject(s)
Adenosine Triphosphate/pharmacology , Arachidonic Acids/blood , Blood Platelets/metabolism , Calcium/pharmacology , Phospholipids/blood , Arachidonic Acid , Blood Platelets/drug effects , Cell Membrane Permeability , Electric Stimulation , Humans , In Vitro Techniques , Kinetics , Ribonucleotides/pharmacology , TritiumABSTRACT
1. Metabolically stable analogues of GTP, e.g. guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta,gamma-imido]triphosphate (pp[NH]pG), enhance the extent of Ca2(+)-dependent secretion of beta-N-acetylglucosaminidase and beta-galactosidase from electropermeabilised human platelets in the presence of less than 5 microM Ca2+. A similar effect is observed on addition either of 1,2-dioctanoin or of GTP in in the presence or absence of thrombin. 2. In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30-40 microM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2(+)-dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2-dioctanoin. 3. The concentration of GTP[S] or pp[NH]pG required to obtain half-maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5-hydroxytryptamine, beta-N-acetylglucosaminidase and beta-galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5-hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of beta-N-acetylglucosaminidase and beta-galactosidase. 4. In the presence of a saturating concentration of GTP[S] marked 5-hydroxytryptamine and beta-N-acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of beta-N-acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C. 5. Guanosine 5'-[beta-thio]diphosphate inhibits enhancement of beta-N-acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone. 6. Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2(+)-dependent platelet lysosomal secretion by activating a guanine-nucleotide-binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.
Subject(s)
Acetylglucosaminidase/metabolism , Blood Platelets/enzymology , Calcium/pharmacology , Galactosidases/metabolism , Guanine Nucleotides/pharmacology , Hexosaminidases/metabolism , Lysosomes/enzymology , beta-Galactosidase/metabolism , Binding Sites , Biotransformation/drug effects , Cell Membrane Permeability/drug effects , Electrochemistry , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/pharmacology , Humans , Thrombin/pharmacologyABSTRACT
Structure-activity studies on a series of analogues of N-(3-methyl-S-(1-pyrrolidinyl carbonyl) butyl)-D-alanine ethyl ester hydrochloride (SC42619) have defined the features of this dipeptide analogue required for observation of thrombin receptor antagonist activity on the human platelet. The affinity for SC42619, and for its structural analogue SC43583 is enhanced by pretreatment of the platelets with chymotrypsin. Endothelial cell prostacyclin (PGI2) synthesis induced by thrombin and trypsin is selectively inhibited by SC42619 provided that prolonged exposure to this antagonist is avoided. However inhibition of PGI2 synthesis by SC42619 is not overcome by increasing the thrombin concentration. The data provide further support for identification of SC42619 and certain of its analogues as selective antagonists at the platelet thrombin receptor but suggest that these compounds may have more complex, and possibly non-selective effects on the endothelial cell.