Herman Van Belle

Kinetics and inhibition of alkaline phosphatases from canine tissues.

Abstract The kinetics and inhibition of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from several tissues of the dog were studied and compared with those of the Escherichia coli enzyme. N -Ethylaminoethanol proved to be the most appropriate buffer. Only small concentrations of magnesium are needed for optimal activity. There is substrate inhibition for all enzymes at concentrations above 1 mM. In the dog neither substrate specificity, nor K m , nor the effect of various inhibitors could differentiate the enzymes from kidney, bone, liver, placenta or mammary tumor whereas the intestinal enzyme was clearly different. The bacterial enzyme differs in many ways from the canine enzymes. The anthelmintic levamisole and one of its analogues, R 8231, were found to be very potent inhibitors of alkaline phosphatase from all tissues except intestine.

R 24 571: A potent inhibitor of calmodulin-activated enzymes

R 24 571, a derivative of the antimycotic miconazole, is a very potent inhibitor of several Ca2+-calmodulin-dependent enzymes. Depending on the concentration of calmodulin, the I50-values range from 0.5 − 1.0 × 10−8 M for brain phosphodiesterase to 1.5 − 3.5 × 10−7 M for erythrocyte Ca2+-ATPase and 5.0 × 10−6 M for phosphorylase b kinase. Being much more potent, and devoid of affinity towards several receptors, R 24 571 is proposed as a more specific and useful tool for studying the involvement of calmodulin in biological processes than the currently used compounds.

Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts

A high-performance liquid chromatography (HPLC) method is described for the separation and quantitation of nucleotides, nucleosides, purine bases, and related compounds in one single run. The separation of a standard mixture of at least 24 components is achieved within 35 min on glass columns (30 cm, 3-mm i.d.) with C-18 reversed-phase particles of 5 micron, and ammonium dihydrogen phosphate (0.15 M, pH 6.00) and a slow linear gradient of methanol/acetonitrile (to 15%) as eluting solvent. The method has been applied to microsamples of different cells and tissues. Samples (2.5 mg dry wt) were cooled in liquid nitrogen, lyophilized, and extracted with 0.6 N perchloric acid. After neutralization with potassium bicarbonate, the extract (20 microliter) was directly injected into the column. To illustrate the wide applicability of the method, representative chromatograms are shown of extracts of biopsies from heart tissue, skeletal muscle, and brain and liver and from hepatocytes, erythrocytes, and yeast cells, under different conditions, known to induce changes in purine metabolism.

MITOCHONDRIAL FUNCTION IN MYOCARDIAL STUNNING

Mitochondrial respiration parameters were studied in mitochondria isolated from normal, ischemic and post-ischemic rabbit hearts. Mitochondrial function was related to tissue content of high energy phosphates (HEP) and cardiac function in the isolated working rabbit heart preparation. It was found that after 10 and 20 mins of global normothermic ischemia followed by 20 mins of Langendorff reperfusion, mitochondrial function and HEP content of the myocardium were not significantly diminished. Myocardial creatine phosphate even showed a significant overshoot as compared to the pre-ischemic condition. When these hearts were allowed to perform work, recovery of cardiac function was incomplete while mitochondrial function and HEP content remained in the normal range. Prolonged ischemia (30 mins) resulted in a significant depression of mitochondrial function and myocardial ATP content during and after ischemia. Recovery of contractile function was severely depressed. These results show that impaired cardiac function after a mild ischemic insult (myocardial stunning) can be associated with near normal mitochondrial function and HEP contents.

Optimization of a high-performance liquid chromatographic method for the determination of nucleosides and their catabolites. Application to cat and rabbit heart perfusates.

A high-performance liquid chromatographic method is described for the separation of nucleosides and related compounds in a single isocratic run. The separation of a standard mixture of at least thirteen compounds is achieved within 15 min on a new type of reversed-phase column 25 cm long, filled with 5-micron particles of Select B. Pre-treatment of the silica particles before silanization has given this type of reversed-phase material unique characteristics for basic and acidic compounds. Chromatograms are shown to compare the effectiveness of the Select B column for the separation of nucleosides with that of other C18 and C8 phases. The separation is achieved with 0.3 M ammonium dihydrogen phosphate (pH 4.00) and a mixture of methanol, acetonitrile and tetrahydrofuran. Detection is carried out with a variable-wavelength UV detector at 254 nm. Sample preparation and the influence of the organic solvents, pH and buffer concentration are described. To illustrate the applicability of the method, representative chromatograms are shown of perfusates of Langendorff preparations and the working hearts of cats and rabbits. Remarkable differences were obtained before and after ischaemia and before and after treatment with a nucleoside transport inhibitor. Baseline separation of cytosine, orotic acid, cytidine, uracil, uric acid, hypoxanthine, xanthine, inosine, guanosine, xanthosine, allopurinol, thymine and adenosine was achieved. The detection limit for these compounds was less than 1 ng per injection.

Kinetics and inhibition of rat and avian alkaline phosphatases

Abstract 1. 1. Rat alkaline phosphatases (orthophosphoric monoester phosphohydrolase, E.C.3.13.1) from liver, bone, kidney, placenta and heart all behave as similar enzymes as far as pH optimum and inhibition are concerned. The liver enzyme showed a distinct substrate specificity while the intestinal enzyme is completely different from all others. 2. 2. Results obtained at rather high substrate concentrations and pH do not necessarily reflect the physiological situation. 3. 3. Levamisole and its analogue, R8231, are potent inhibitors of all rat alkaline phosphatases except the intestinal one. 4. 4. These compounds could be successfully used for the quantitation of the important intestinal fraction of rat serum alkaline phosphatase. 5. 5. The alkaline phosphatases from chicken liver, bone and kidney are much less sensitive to levamisole or R8231 than the corresponding isoenzymes from mammalia. 6. 6. The alkaline phosphatase activity in chicken serum is inhibited by R8231 only in the adult animals. No inhibition was observed in two weeks old chicken where the intrinsic activity is much higher.

Inhibition of nucleoside transport by a new series of compounds related to lidoflazine and mioflazine

A new series of compounds related to the nucleoside transport inhibitors, lidoflazine and mioflazine, is introduced. The influence of these derivatives on nucleoside-specific transport proteins was studied in two ways. First, a rapid, non-radioactive assay was developed for the screening of this type of material for actual transport inhibition in human erythrocytes. The method is based on the dose-dependent reversal of the inhibition of inorganic phosphate release induced by inosine when human erythrocytes are suspended in a phosphate-free medium. It enables the estimation of the potency and specificity of this new series of nucleoside transport inhibitors, most of which are highly active (EC50 values as low as 13 nM). Second, the displacement of a radiolabeled transport inhibitor, [3H]nitrobenzylthioinosine, was examined. All compounds were capable of displacing specific [3H]nitrobenzylthioinosine binding to crude and solubilized plasma membranes of calf lung tissue, displaying affinities in the nanomolar range. Pseudo-Hill coefficients derived from the shape of the displacement curves were significantly greater than unity for most derivatives, in contrast to values of approximately unity obtained for dipyridamole and analogs. These findings were incorporated in a mathematical model describing the interaction of mioflazine analogs with the transport protein, suggesting that one molecule of mioflazine is capable of displacing two or more molecules of [3H]nitrobenzylthioinosine at a time. The consequences of this model regarding the nature of the transport protein are discussed.

Cardioprotective effects of nucleoside transport inhibition in rabbit hearts

The cardioprotective effects of a nucleoside transport inhibitor, R75231, were investigated in the isolated rabbit heart. The hearts were subjected to 20 minutes of global normothermic ischemia followed by reperfusion. Before ischemia either solvent (group 1), 5 mumol/L of adenosine (group 2), or 0.64 mg/L R75231 (group 3) was added to the perfusate. Preischemic hemodynamics were not changed by treatment, except for an increase in coronary flow in the adenosine group (126% of control; p less than 0.05). Upon reperfusion, coronary flow was depressed in the controls (72% of the preischemic control values), increased in the adenosine group (113%) and unchanged in the R75231 group (89%). Functional recovery was significantly better in the adenosine group as well as in the R75231 group as compared with the controls (p less than 0.05). Cardiac output was 74% of the preischemic control value in the R75231 group, 67% in the adenosine group, and only 38% in the controls. Analysis of the coronary effluent after reperfusion showed a significant inhibition of rapid release of purines and a reverse of the adenosine/inosine ratio in the R75231 group as compared with the others. We conclude that R75231 has a cardioprotective effect that is probably related to accumulation of endogenous adenosine.

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.

The disappearance of adenosine in blood effect of lidoflazine and other drugs

Abstract Of 23 different drugs, tested at 1 × 10 −6 M, only the three specific coronary vasodilators, lidoflazine, dipyridamole and hexobendine markedly decreased the rate of uptake of addedadenosine by erythrocytes in whole human blood. The inhibitory action of lidoflazine on adenosine permeability of human erythrocyte membranes was much more resistant to repeated washes than that of the two other inhibitors, suggesting the formation of a more stable complex with some unknown erythrocytic membrane component. Plasma or lysed blood adenosine deaminase activity was unaffected by lidoflazine at 1 × 10 −6 M, in man and in dogs. The inhibitory effect of lidoflazine on the rate of disappearance of added adenosine from dog whole blood was due to a permeability decreasing action on the platelet membranes; the erythrocytes of the dog were, suprisingly unaffected by the drug. The effect of lidoflazine on the rate of disappearance of added adenosine in whole blood was found to be species dependent. At 10 −7 M the drug had a pronounced rate decreasing effect in the chicken, the rabbit, the pig, the human and the dog and a more moderate effect in the guinea-pig. It was virtually ineffective, at 10 t-7 M, in the cat, the sheep and the rat. The potency of the drug was directly correlated with the adenosine uptake capacity of the blood corpuscules ( p = 0.002) and inversely correlated with the plasma denosine deaminase activity ( p = 0.008). The adenosine uptake capacity, deaminase activity and lidoflazine sensitivity were qualitatively and quantitatively strikingly similar in the blood of the human and of the pig, suggesting that the pig is a better experimental model for studying the coronary effects of lidoflazine than any one of the seven other speicies studied.