ABSTRACT
To investigate whether diacylglycerol (DAG) has a role in reversible platelet aggregation induced by low concentrations of platelet-activating factor (PAF), we attempted to use the DAG kinase inhibitor, R59022, to prevent rapid conversion of DAG to phosphatidic acid. However, we found that R59022 inhibited the binding of [3H]PAF to human and rabbit platelets and to rabbit platelet membranes. We then investigated whether exogenous, cell-penetrating DAGs (1,2-dihexanoyl-sn-glycerol (DHG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG)) act synergistically with low concentrations of PAF that alone induce only reversible aggregation. Platelets were isolated and labeled with [14C]serotonin. DHG (25-75 microM) caused slow, weak aggregation and some release of [14C]serotonin with human, but not rabbit, platelets. OAG (25-75 microM) did not aggregate either species' platelets. Phosphorylation of pleckstrin by DHG was more transient in rabbit platelets than previously observed with human platelets. Both DHG and OAG synergistically potentiated PAF-induced aggregation of human platelets, but, paradoxically, concurrently inhibited the PAF-induced increase in intracellular Ca2+ ([Ca2+]i): potentiation decreased upon incubation with DAGs before PAF addition. In contrast, DHG strongly inhibited PAF-induced aggregation of rabbit platelets; inhibition decreased upon preincubation. OAG, added with PAF, slightly potentiated aggregation of rabbit platelets: upon preincubation, OAG progressively inhibited. Effects of DHG and OAG on PAF-induced increases in [Ca2+]i in rabbit platelets followed a similar pattern; thus, with rabbit platelets, inhibition of the [Ca2+]i increase may at least partially account for inhibition of PAF-induced aggregation by exogenous DAGs. Results with human platelets are consistent with stimulation of protein kinase C by DAGs, and then metabolism of DAGs and/or negative feedback by DAGs, but results with rabbit platelets indicate both an unexpected species difference and a difference between the effects of DHG and OAG on PAF-induced platelet aggregation.
Subject(s)
Blood Platelets/drug effects , Diglycerides/pharmacology , Platelet Activating Factor/pharmacology , Animals , Blood Platelets/metabolism , Cell Membrane Permeability/drug effects , Diacylglycerol Kinase , Diglycerides/blood , Diglycerides/pharmacokinetics , Drug Synergism , Enzyme Inhibitors/pharmacology , Humans , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Platelet Activating Factor/antagonists & inhibitors , Platelet Activating Factor/metabolism , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Pyrimidinones/pharmacology , Rabbits , Thiazoles/pharmacologyABSTRACT
Probenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.
Subject(s)
Ion Channels/antagonists & inhibitors , Penicillin G/pharmacology , Penicillins/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Probenecid/pharmacology , Uricosuric Agents/pharmacology , Calcium/blood , Drug Synergism , Humans , In Vitro Techniques , Serotonin/blood , Whole Blood Coagulation TimeABSTRACT
The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although the reason for this effect is unknown, it is unlikely to involve inhibition of phospholipase C, since formation of diacylglycerol appears to have little involvement in the primary phase of ADP-induced aggregation.
Subject(s)
Adenosine Diphosphate/pharmacology , Platelet Aggregation/drug effects , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Alkaloids/pharmacology , Blood Platelets/drug effects , Blood Platelets/metabolism , Diacylglycerol Kinase , Diglycerides/pharmacology , Enzyme Activation/drug effects , Ethers, Cyclic/pharmacology , Humans , Okadaic Acid , Phosphotransferases/antagonists & inhibitors , Pyrimidinones/pharmacology , Serotonin/blood , Staurosporine , Thiazoles/pharmacology , Thrombin/pharmacologyABSTRACT
1. Variations in the concentration of Ca2+ [Ca2+] in the suspending medium have different effects on the responses of human and rabbit platelets to collagen. 2. When rabbit platelets are stimulated with a low concentration of collagen (0.5 micrograms/ml), aggregation, release of granule contents, and formation of thromboxane are maximal when the suspending medium contains [Ca2+] in the physiological range (0.5-2.0 mM), and very slight in a medium with no added Ca2+. 3. In contrast, human platelets respond most strongly when the suspending medium contains no added Ca2+ [( Ca2+] approx. 20 microM); this is attributable to the enhanced formation of thromboxane A2 (TXA2) upon close platelet-to-platelet contact in this medium. 4. When TXA2 formation is blocked by inhibition of cyclo-oxygenase with aspirin or indomethacin, rabbit platelet aggregation and release in response to 1.25-10 micrograms/ml collagen is also maximal at [Ca2+] of 0.5-2.0 mM and least at 20 microM; human platelets do not aggregate and the extent of release is relatively independent of [Ca2+]. 5. In 1 mM [Ca2+], use of apyrase and/or ketanserin with rabbit platelets in which TXA2 formation is blocked shows that released ADP and serotonin make large contributions to aggregation and release in response to high concentrations of collagen; human platelet aggregation is largely dependent on TXA2. 6. Use of fura-2-loaded platelets shows that the collagen-induced rise in cytosolic [Ca2+] is only slightly inhibited by aspirin or indomethacin in rabbit platelets, but almost completely inhibited in human platelets. 7. Responses of rabbit platelets to collagen are less dependent on TXA2 than those of human platelets. Released ADP and serotonin make major contributions to the responses of rabbit platelets to collagen.
Subject(s)
Blood Platelets/metabolism , Calcium/metabolism , Collagen/physiology , Animals , Aspirin/pharmacology , Cell Degranulation , Cyclooxygenase Inhibitors , Cytosol/metabolism , Humans , In Vitro Techniques , Indomethacin/pharmacology , Ketanserin/pharmacology , Kinetics , Rabbits , Thromboxane A2/biosynthesisABSTRACT
Elderly alcoholics represent an increasing number of older Americans caught in the trappings of epidemiologic and treatment problems. This paper reviews some current dimensions along both problems in light of the unresolved statistics of addiction. Reductions in the federal budget and public services lend contrast to the proportion of cases associated with socioeconomic distress, and mental illness. In the second part on treatment considerations, advances in behavioral pharmacology and behavioral psychotherapy are briefly discussed. The holistic models of behavioral medicine and field-integrative therapy are indicated as possible directives for a multicausal assessment and community intervention.
Subject(s)
Alcoholism/rehabilitation , Aged , Alcoholism/epidemiology , Alcoholism/psychology , Behavior Therapy , Combined Modality Therapy , Cross-Sectional Studies , Humans , Poverty , Public Policy , Risk , United StatesSubject(s)
Assertiveness , Substance-Related Disorders/psychology , Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Reference ValuesSubject(s)
Behavior Therapy/instrumentation , Enuresis/therapy , Adolescent , Child , Enuresis/psychology , HumansSubject(s)
Assertiveness , Disabled Persons/psychology , Psychodrama , Role Playing , Aged , Female , HumansSubject(s)
Aged/psychology , Assertiveness , Behavior Therapy , Personality Assessment , Disabled Persons/psychology , Female , HumansSubject(s)
Fetal Alcohol Spectrum Disorders/psychology , Abnormalities, Multiple/chemically induced , Alcoholism/epidemiology , Animals , Female , Fetal Growth Retardation/chemically induced , Hippocampus/drug effects , Humans , Hyperkinesis/etiology , Learning Disabilities/chemically induced , Male , Pregnancy , Rats , United StatesSubject(s)
Assertiveness , Behavior Therapy/methods , Blindness/rehabilitation , Adolescent , Adult , HumansABSTRACT
It has been shown earlier that the interactions of the isolated rat peritoneal mast cells with cationic protein from rabbit neutrophil lysosomes (band 2 protein) can be studied using anilinonaphthalene sulfonate (ANS) as a fluorescent probe. In the present communication, binding of ANS dye to the mast cells interacting of histamine release by metabolic inhibitors was found to have no effect on enhancement of ANS fluorescence. On the other hand, inhibition of histamine release at high concentration of Ca2+ (14.4 mM) was accompanied by the decrease in enhance fluorescence. In the presence of 7.2 mM of Sr2+, the release of histamine was enhanced with small but significant increase in ANS fluorescence. The cells heated to 42 degrees C partially lost their capacity to release histamine without the loss of enhanced fluorescence. The mast cells interacting with B2 at 10 degrees C for various time intervals showed time-dependent loss in histamine releasing capacity with concomitant loss in enhanced fluorescence. These studies suggest that the enhancement of ANS fluorescence is associated with the early events of the cell membrane caused by interaction of B2 with the cells. The extracellular cations significantly influence this early event.
Subject(s)
Antimetabolites/pharmacology , Histamine Release/drug effects , Lysosomes/metabolism , Mast Cells/metabolism , Proteins/metabolism , Anilino Naphthalenesulfonates , Animals , Calcium/pharmacology , Cations , In Vitro Techniques , Male , Rabbits , Rats , Spectrometry, FluorescenceABSTRACT
Histamine release from rat mast cells induced by cationic protein (band 2) from rabbit neutrophil lysosomes occurs in Ca++-deficient medium. At higher concentrations of Ca++ the release is inhibited. Strontium not only supports, but also enhances the release of histamine in the absence of Ca++. Progressive enhancement of release occurs between 1.8 and 14.4 mM Sr++. The release of histamine from mast cells, activated at low temperature (0-4 degrees C) in the presence of 14.4 mM Ca++ and then washed prior to incubation at 37 degrees C, is inhibited. However, if phosphatidyl serine (PS) (10 microgram) is present with 14.4 mM Ca++, the inhibition is reversed. There is also inhibition of release when cells, activated in the presence of 1.8 mM Ca++, are incubated in the second stage with 14.4 mM Ca++, but this inhibition is less pronounced than when the 14.4 mM Ca++ is in the activation stage. PS enhances the release in the presence of both Ca++ and Sr++. The presence of PS in the activation stage enhances the release, but there is no significant enhancement when cells activated in the absence of PS are washed and incubated in the presence of PS. This suggests that PS enhancement of histamine release occurs at the activation stage, probably through the efficient delivery of calcium to the membrane sites, thereby increasing the efficacy of the membrane perturbation by band a protein.