Metabolic aspects of the secretion of stored compounds from blood platelets. IV. Effects of ionophore X537A on washed platelets.

1. X537A at concentrations below 10 muM can liberate platelet serotonin from washed human platelets without inducing the platelet release reaction. Up to 100% of serotonin preabsorbed by the platelets can be liberated before initiation of the release reaction. 2. Concentrations of X537A above 10muM initiate the platelet release reaction, with a maximum release of adenine nucleotides and platelet factor 4 antigen comparable to that obtained with 1.25 units thrombin/ml. 3. The changes in ATP metabolism at the concentration necessary for X537A-induced release are more profound than those in platelets exposed to concentrations of thrombin or A23187 giving the same degree of release, and approach those seen with high concentrations of A23187. At concentrations where serotonin is liberated but no adenine nucleotide or platelet factor 4 antigen is released, short time incubation causes no change in the level of metabolic ATP.

Metabolic aspects of the secretion of stored compounds from blood platelets. V. Effect of ionophore A23187 on washed platelets.

1. The A23187-induced secretion of preabsorbed serotonin from human blood platelets at 37 degrees C is studied. Preincubation at the same temperature before the addition of ionophore is necessary for maximal release induction. When total incubation time is kept constant, longer time with ionophore results in a smaller decrease in the level of metabolic ATP and increase in metabolic ATP and increase in metabolic IMP. This coincides with the reduction in secretion, but statistical treatment of the results suggests that the reduced secretion only partially explains the reduced drop in metabolic ATP, and that therefore a resynthesis of metabolic ATP from IMP may have taken place. 2. In some experiments induction of secretion takes place over a very narrow range of ionophore concentration. 3. When K+ substitutes for Na+ in the extracellular medium, the need for preincubation for maximal secretion becomes less evident, and at times is abolished, while there is still a significant increase in the metabolic ATP level by prolonged incubation with ionophore. 4. A reduction in secretion is observed with metabolic blockers when the ionophore is added after preincubation, but to a much less degree than when secretion is induced by thrombin, in spite of a great reduction in the level of metabolic ATP. This may partly be explained by the increase in secretion induction by A23187 in the presence of inhibitors when the ionophore is added in the cold, suggesting that the inhibitor may cause "weakening" of the platelets' "resistance" to induction of secretion by ionophore. 5. When the effect of Ca2+ and of Mg2+ on the level of intermediates of the TP leads to hypoxanthine conversion is studied, it is evident that the addition of Ca2+ causes enhanced IMP accumulation and a reduction in the level of inosine plus hypoxanthine, while Mg2+ has the opposite effect. This suggests that the two metals affect the enzymes of the IMP leads to hypoxanthine conversion differently. 6. Indomethacin inhibits secretion induced by A23187, suggesting that prostaglandin intermediates may amplify the ionophore-induced release. The adenine nucleotide metabolism is not affected. 7. The results indicate that there is an indirect, rather than direct, link between the major metabolic changes and the secretion induced by A23187, but that the ionophore may cause intracellular changes which are not connected to its effect as release inducer.

Effects of oleate and insulin on glucose uptake, oxidation, and glucose transporter proteins in rat adipocytes.

We examined effects of Na oleate on glucose uptake, glucose transporter protein concentrations, and glucose oxidation in isolated adipocytes from fed rats. Na oleate increased basel 14C-glucose uptake in a dose-dependent manner (+42% with 1.0 mM, +79% with 2.8 mM Na oleate), but had no statistically significant effect on insulin-stimulated glucose uptake. Insulin (100 nM) resulted in a redistribution of GLUT4 protein concentration from the LDM fraction (-42%) to the PM fraction (+266%) but did not affect the distribution of GLUT1. Na oleate had no effect on basal or insulin-stimulated concentrations of GLUT1 or GLUT4 proteins in the PM or LDM fractions. Na oleate (2.8 mM) had no statistically significant effect on basal glucose oxidation, but inhibited insulin-stimulated glucose oxidation by 48% (P less than 0.01). In summary, Na oleate inhibited insulin-stimulated glucose oxidation and stimulated basal glucose uptake in isolated adipocytes without affecting PM or LDM distribution of GLUT1 or GLUT4 proteins. We conclude that the stimulatory effect of Na oleate on basal glucose uptake in adipocytes may be mediated by changes in the intrinsic activity of the glucose transporters.

Phosphorus labeling of proteins and phospholipids in intact platelets in response to pH 5.3.

Previous studies had shown that when gel-filtered or washed human platelets were incubated at pH 5.3, the cells secreted their granule-stored materials suggesting that low pH can act as a platelet activator. We determined here whether the effects of low pH on platelet protein phosphorylation and on platelet lipid metabolism were consistent with this view. When washed human platelets were incubated for 20 min at pH 5.3 and electrophoresed on SDS-PAGE, there was a great increase in 32P-label in the 20,000 and 47,000 dalton protein bands. There was also an increase in the labeling of phosphatidic acid and a small decrease in phosphatidyl inositol. When the platelets were returned to pH 7.6, the 32P labeling of the 20,000 and 47,000 dalton bands was greatly reduced, and that of phosphatidic acid reduced to the control value, while the labeling of phosphatidyl inositol was increased above control. Incubation at pH 5.3 for 60 min gave the same pattern, but return to pH 7.6 resulted in only partial reversal of labeling of the two protein bands and little decrease in the label associated with phosphatidic acid, but the radioactivity in phosphatidyl inositol was greatly increased. The changes in the 32P-labeling of phospholipids and proteins after incubation of platelets at pH 5.3 may reflect an increase in cytoplasmic Ca++ resulting from leakage of Ca++ from intracellular storage sites, a process which becomes irreversible after longer time exposure to the low pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of thrombin-induced secretion from platelets by chlortetracycline and its analogs.

Chlortetracycline (CTC) (1.0 mM) blocks platelet secretion after a few seconds preincubation. The amount needed for inhibition can be reduced relative to time of preincubation. 50 micro M CTC. Two tetracycline analogs, anhydrotetracycline and demeclocycline (DMC), have different solubility properties in nonionic medium, but inhibit secretion at the same concentration, with little effect on the metabolic ATP level. The results suggest that CTC and its analogs do not inhibit platelet function by acting as metabolic inhibitors. While CTC causes increased leakage of cytoplasmic content at the concentrations where secretion is blocked more than 90%, DMC doses not cause leakage even at much higher concentration, so that there seems to be no connection between the induction of leakage (i.e. the membrane-active properties) and the inhibitory effect of the drugs.

Effects of 8-(N, N-diethylamino)ocytl 3, 4, 5-trimethoxybenzoate HCl (TMB-8) on washed blood platelets with special reference to a specific loss of serotonin.

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (TMB-8) has been used to indicate involvement of Ca++ in platelet activities, but possible other effects have not been investigated. This study investigates TMB-8-induced leakage of cytoplasmic content and specific loss of one granule constituent, serotonin from washed platelets. TMB-8 concentrations above 0.5 mM block secretion initially and induce leakage after short time incubation. TMB-8 (0.15 mM) enhances a slow release of serotonin, but inhibits a slow release of low affinity platelet factor 4 antigen which take place when washed platelets are incubated at pH 7.4 and 37 degree C. These effects should be taken into consideration when TMB-8 is used as a calcium antagonist in platelets.

Protease inhibitors in Haementeria leech species.

Inhibitors, of trypsin, plasmin, alpha-chymotrypsin and granulocyte elastase were demonstrated in salivary gland extracts from two species of leeches. Haementeria ghilianii and Haementeria officinalis. Preliminary fractionation of salivary gland extracts from Haementeria ghilianii allowed separation of protease inhibitors from hementin a fibrinogenolytic blood anticoagulant. It was found that the anticoagulant activity resided only in hementin-containing fractions and did not parallel protease inhibitory activity.

15-Deoxy-delta(12,14) prostaglandin J2: a putative endogenous promoter of adipogenesis suppresses the ob gene.

Leptin is considered a key factor in the regulation of appetite and energy expenditure, but little is known about the control of its synthesis and release. Thiazolidinediones (TZDs) have recently been shown to downregulate leptin expression, and it has been speculated that downregulation of the ob gene occurs through activation of the transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma). However, there are no studies using an endogenous PPARgamma ligand. We examined the effect of 15-deoxy-delta(12,14) prostaglandin J2 (15d-PGJ2), a putative natural ligand of PPARgamma, on ob gene expression in fully differentiated 3T3-L1 adipocytes and compared its effect with that of two other PPARgamma activators, the TZD troglitazone (Trog) and indomethacin (Indo). 15d-PGJ2, Trog, and Indo all inhibited leptin expression at concentrations at which they activate PPARgamma. The inhibition of leptin expression of PPARgamma activators was surprising, since PPARgamma is known to induce adipogenesis during which the ob gene is expressed. To address the possibility that PPARgamma plays different roles before and after the induction of adipogenesis, we examined the effects of the three PPARgamma ligands on the expression of leptin and the glucose transporter protein GLUT4, both of which are expressed during differentiation of 3T3-L1 preadipocytes to adipocytes. In the absence of PPARgamma ligands, leptin and GLUT4 synthesis increased from day 3 to day 9 or 10 during differentiation. However, in the presence of any of the three PPARgamma ligands, GLUT4 expression was unaffected, while ob gene expression was inhibited. We hypothesize that PPARgamma may be essential for induction of adipocyte differentiation but then needs to be inactivated to allow expression of the ob gene.