H. Van Belle

12-O-Tetradecanoylphorbol 13-acetate stimulates inositol lipid phosphorylation in intact human platelets

Potyphosphoinositide Phorbol ester Protein kinase C

Uptake and deamination of adenosine by blood. Species differences, effect of pH, ions, temperature and metabolic inhibitors.

Abstract 1. 1. The uptake and/or deamination of adenosine has been measured in continuo in blood from 12 different species. 2. 2. In total blood, the highest uptake was found in chicken and rabbit, less in human and pig, much less in dog, rat, guinea pig, sheep, goat and cat and almost negligible in cow and horse. 3. 3. Adenosine is deaminated to a large extent by the plasma in sheep, goat, cow and cat. In the other species, the erythrocytes are mainly involved in uptake and deamination except in guinea pig and dog in which the platelets are largely responsible for the disappearance of adenosine. 4. 4. In human and canine blood, the inactivation of adenosine is highly dependent on temperature but rather insensitive to changes in pH between 6 and 8 or to ionic environment. 5. 5. In human blood, metabolic inhibitors such as KCN, NaF, malonate, 2,4-din itrophenol or ouabain do not affect adenosine uptake or adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) activity. p -Chloromercuribenzoate inhibits the deaminase activity without changing the uptake, whereas N -ethylmaleimide exhibits the opposite effects.

New and sensitive reaction for automatic determination of inorganic phosphate and its application to serum

Abstract Based on a new reaction—complex formation, with spectral shift, between phosphomolybdate and the dye, Methyl Green 00—an automatic method has been developed for the determination of inorganic phosphate. The advantages of the method are its very high sensitivity when compared with the existing methods, its extreme simplicity and the very short reaction time (2.5 min) under relatively mild conditions (1.25 N HCl). The hydrolysis of labile phosphates was found negligible. The reproducibility of the method is excellent (relative S.D. = ±0.91%). The method has been applied to the automatic determination of inorganic phosphate in serum. The higher sensitivity results in the uptake of much less sample and the simplicity makes serum phosphate determination less troublesome and less time consuming. In ten consecutive analyses of a serum sample containing 5.59 mg % PO4P the standard deviation was found to be ±0.054. The analysis of 6 different control sera showed an excellent accuracy. Phosphate added to these sera could be completely recovered (99.8–101.5%).

Comparative Pharmacology of Nucleoside Transport Inhibitors

Abstract The few existing nucleoside transport inhibitors are almost equipotent on the transporter in washed human erythrocytes. Large differences exist however in tightness of binding and activity in the presence of human plasma, as well as in ex vivo inhibition and duration of action when given intravenously or orally. Examples are presented of the marked effect of nucleoside transport inhibition on adenosine accumulation in ischemic myocardium. That may explain the remarkable cardioprotection in several models when a nucleoside transport inhibitor with an appropriate pharmacokinetic profile (such as R 75 231) is applied.

Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humans. Implications for its future role in cardioprotection.

In 12 healthy male volunteers (27-53 yr), a placebo-controlled randomized double blind cross-over trial was performed to study the effect of the intravenous injection of 0.25, 0.5, 1, 2, 4, and 6 mg draflazine (a selective nucleoside transport inhibitor) on hemodynamic and neurohumoral parameters and ex vivo nucleoside transport inhibition. We hypothesized that an intravenous draflazine dosage without effect on hemodynamic and neurohumoral parameters would still be able to augment the forearm vasodilator response to intraarterially infused adenosine. Heart rate (electrocardiography), systolic blood pressure (Dinamap 1846 SX; Critikon, Portanje Electronica BV, Utrecht, The Netherlands) plasma norepinephrine and epinephrine increased dose-dependently and could almost totally be abolished by caffeine pretreatment indicating the involvement of adenosine receptors. Draflazine did not affect forearm blood flow (venous occlusion plethysmography). Intravenous injection of 0.5 mg draflazine did not affect any of the measured hemodynamic parameters but still induced a significant ex vivo nucleoside-transport inhibition of 31.5 +/- 4.1% (P < 0.05 vs placebo). In a subgroup of 10 subjects the brachial artery was cannulated to infuse adenosine (0.15, 0.5, 1.5, 5, 15, and 50 micrograms/100 ml forearm per min) before and after intravenous injection of 0.5 mg draflazine. Forearm blood flow amounted 1.9 +/- 0.3 ml/100 ml forearm per min for placebo and 1.8 +/- 0.2, 2.0 +/- 0.3, 3.8 +/- 0.9, 6.3 +/- 1.2, 11.3 +/- 2.2, and 19.3 +/- 3.9 ml/100 ml forearm per min for the six incremental adenosine dosages, respectively. After the intravenous draflazine infusion, these values were 1.6 +/- 0.2 ml/100 ml forearm per min for placebo and 2.1 +/- 0.3, 3.3 +/- 0.6, 5.8 +/- 1.1, 6.9 +/- 1.4, 14.4 +/- 2.9, and 23.5 +/- 4.0 ml/100 ml forearm per min, respectively (Friedman ANOVA: P < 0.05 before vs after draflazine infusion). In conclusion, a 30-50% inhibition of adenosine transport significantly augments the forearm vasodilator response to adenosine without significant systemic effects. These results suggest that draflazine is a feasible tool to potentiate adenosine-mediated cardioprotection in man.

Sleep improvement in dogs after oral administration of mioflazine, a nucleoside transport inhibitor.

Mioflazine, a nucleoside transport inhibitor, was given PO to dogs at doses of 0.04–10 mg/kg. Sixteen hour polygraphic sleep recordings were made and analysis and sleep stage classification was done by computer. Mioflazine decreased wakefulness and increased slow wave sleep, but did not affect the latencies of either REM sleep or slow wave sleep. This increased sleep was due to an increase in the number of light and deep slow wave sleep epochs. The effect lasted for about 8 h. The decreased wakefulness and increased slow wave sleep could be antagonized by the adenosine antagonist caffeine (2.5 and 10 mg/kg, PO); however, there was not a pure antagonistic effect. It might be that the enhancement of slow wave sleep is due to an activation of brain adenosine receptors. This is the first report of a drug acting on adenosine that given orally improves sleep. Mioflazine might be the prototype of substances worth considering for the treatment of a variety of sleep disorders.

Stimulation by serotonin of 40 kDa and 20 kDa protein phosphorylation in human platelets

In human platelets, serotonin is known to induce a shape change followed by (reversible) aggregation. Recently, it was found that the amine triggers the elevation of cytosolic free calcium and activates phospholipase C. On stimulation of human platelets with serotonin we found an immediate increase in protein kinase C activity, phosphorylating its 40 kDa substrate protein. A 20 kDa protein, most likely the myosin light chain, was phosphorylated to the same extent. Ketanserin, a highly selective serotonin‐S2 antagonist inhibited both phosphorylation processes at subnanomolar concentrations.

Agents that elevate platelet cAMP stimulate the formation of phosphatidylinositol 4-phosphate in intact human platelets.

The present study investigates the effect of compounds that are known to elevate cAMP on the phospholipid metabolism of platelets. Prostaglandin E1, forskolin and isobutylmethylxanthine induce an increase in [32P]‐phosphatidylinositol 4‐phosphate (PIP) in platelets prelabelled with [32P]orthophosphate. Possible roles of this phenomenon are discussed in view of the inhibitory effect of cAMP elevation on platelet activation.

The effect of calcium on mitochondrial contact sites: a study on isolated rat hearts.

Mitochondrial contact sites are dynamic structures created by fusion of the inner and outer mitochondrial membranes. Stimulation of the metabolism results in an increase of the number of contact sites. Functionally, it is shown that mitochondrial creatine kinase (Mi-CK) is active in contact sites and therefore, Mi-CK cytochemistry was performed (using a tetrazolium salt) to improve the visibility of the contact sites. As calcium is involved as an intracellular messenger of hormonal stimulation, the effect of increasing extracellular calcium concentrations on the number of contact sites was investigated. Therefore, isolated rat hearts were perfused with Krebs-Henseleit buffers differing in their calcium content. During the perfusions the heart function was evaluated and at the end of each experiment, the hearts were processed for Mi CK cytochemistry and the number of contact sites was expressed as the ratio of surface densities contact sites to mitochondrial membranes (Ss). At 2.2 mM calcium perfusion, the physiological parameters and the Ss reached a maximum. This was in contrast to the 0.6 and the 3.6 mM of calcium perfusions whereby both the physiological values and the Ss were decreased. Treatment with noradrenaline in vivo, as was done in previous studies or perfusion with 2.2 mM of calcium ends up with similar values for Ss. From these results, it could be suggested that there might be a link between calcium, heart function and the formation of Mi CK active contact sites.

1-Oleoyl-2-acetyl-glycerol (OAG) stimulates the formation of phosphatydylinositol 4-phosphate in intact human platelets

Diacylglycerol (DAG) is one of the primary products formed upon activation of platelets with stimuli that induce inositol lipid turnover. Its synthetic analog, 1-oleoyl-2-acetyl-glycerol (OAG) is often used as a tool for studying the involvement of the lipid in platelet activation. We found that OAG induces a concomitant increase in [32P]-incorporation in phosphatidylinositol 4-phosphate (PIP) and in the 40K protein, the endogenous substrate for protein kinase C in human platelets. It is hypothesized that in receptor mediated platelet activation a metabolic link might exist between both processes.