[Effect of instenon on the uterine contractile activity]. / Vliianie instenona na sokratitel'nuiu deiatel'nost' matki v éksperimente.

The effect of instenon upon the uterine contractile activity was studied in pregnant rats. The drug administration at a dose resulted in significant suppression of the myometrium contractility. The effect was not decreased when instenon was injected against the background of oxytocin. On the other hand, the concurrent injection of oxytocin upon a single or long-term administration of instenon produced a pronounced stimulating action on the myometrium contractility.

Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis.

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

Adenosine formation and release by embryonic chick neurons and glia in cell culture.

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.

Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells.

Adenosine formation and release was examined in 48 hr old primary cultures of chick ventricular myocytes. Dilazep greater than hexobendine greater than dipyridamole inhibit incorporation of adenosine into chick embryonic heart cellular nucleotides in a concentration dependent manner. A combination of 30 mM 2-deoxyglucose and 2 micrograms of oligomycin/ml reduces the ATP content of the cells by 71% in 10 min. This change is accompanied by an increase in total adenosine concentration of 3.4 nmoles/10(7) cells in 10 min. Although the ATP concentration is not altered during hypoxia (95%N2/5%CO2), adenosine concentration increases by 0.52 nmoles/10(7) cells in 30 min. When nucleoside incorporation is inhibited by 85-90% by dipyridamole, dilazep or hexobendine, efflux of adenosine decreases by 70-90%, and 60-90% of the newly formed adenosine is trapped inside the cells compared to 10% in the absence of the transport inhibitors. alpha, beta -Methylene ADP inhibits the ecto 5'-nucleotidase activity by 91 +/- 6% but does not inhibit adenosine formation or alter its distribution between cells and medium, thus ruling out the involvement of this enzyme in adenosine formation. We conclude that adenosine is formed intracellularly during 2-deoxyglucose and oligomycin-induced ATP degradation and during hypoxia and that the nucleoside is released via the symmetric nucleoside transporter.

Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

Inhibition of nucleoside and nucleobase transport and nitrobenzylthioinosine binding by dilazep and hexobendine.

The transport of 500 microM uridine by human erythrocytes and S49, P388 and L1210 mouse leukemia cells, Chinese hamster ovary (CHO) cells and Novikoff rat hepatoma cells was inhibited strongly by dilazep and hexobendine with similar concentration dependence, but the sensitivity of transport in the various cell types varied greatly; IC50 values ranged from 5-30 nM for human erythrocytes and S49 and P388 cells to greater than 1 microM for CHO and Novikoff cells. The binding of nitrobenzylthioinosine (NBTI) to high-affinity sites on these cells (Kd approximately equal to 0.5 nM) was inhibited by hexobendine and dilazep in a similar pattern. Furthermore, these drugs, just as dipyridamole and papaverine, inhibited the dissociation of NBTI from high-affinity binding sites but only at concentrations 10-100 times higher than those inhibiting uridine transport. In contrast, high uridine concentrations (greater than 2 mM) accelerated the dissociation of NBTI. Dilazep also inhibited the transport of hypoxanthine, but only in those cell lines whose transporter is sensitive to inhibition by uridine and dipyridamole. Adenine transport was not inhibited significantly by dilazep in any of the cell lines tested, except for a slight inhibition in Novikoff cells. [14C]Hexobendine equilibrated across the plasma membrane in human erythrocytes within 2 sec of incubation at 25 degrees, but accumulated to 6-10 times the extracellular concentration in cells of the various cultured lines. Uptake was not affected by high concentrations of uridine, NBTI or dipyridamole. Hexobendine inhibited the growth of various cell lines to a lesser extent (IC50 = greater than or equal to 100 microM) than dipyridamole (IC50 = 15-40 microM). At 40 microM, however, it completely inhibited the growth of S49 cells that had been made nucleoside dependent by treatment with methotrexate or pyrazofurin.

Effects of adenosine uptake blockers and adenosine on evoked potentials of guinea-pig olfactory cortex.

The olfactory cortex slice preparation from guinea-pig has been used to test compounds which inhibit the cellular uptake of adenosine. The uptake inhibitors dipyridamole (0.1-10 mumol/l), dilazep (1-10 mumol/l) nitrobenzylthioguanosine (1-10 mumol/l), nitrobenzylthioinosine (0.1-5 mumol/l), and hexobendine (1-100 mumol/l) increased the potency of adenosine (0.1-30 mumol/l) by up to 5-fold but did not potentiate cyclohexyladenosine (0.01-10 mumol/l). The benzodiazepine, diazepam (1 mumol/l) slightly increased the potency of adenosine (by 1.7-fold) whereas flurazepam (3 mumol/l) had no effect, suggesting that inhibition of adenosine uptake is probably not the major therapeutic action of these compounds. The uptake inhibitors depressed the amplitude of the monosynaptic epsp when added alone, an effect reversed by adenosine deaminase (1 unit/ml) whereas the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (10 mumol/l) had no effect on adenosine action. These results show that in this preparation (a) adenosine action is attenuated by an uptake mechanism and (b) endogenous adenosine release normally has no apparent effects on synaptic transmission at low stimulus rates. Nitrobenzylthioinosine and nitrobenzylthioguanosine are probably the best uptake blockers.

Characterization of the adenosine receptor responsible for the inhibition of histamine and SRS-A release from human lung fragments.

The inhibitory effects of a range of natural and synthetic derivatives of adenosine on the antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung has been studied. The nucleotides ATP, ADP and AMP appear to act by being converted to adenosine. The rank order of inhibitory potency of the synthetic analogues indicates that these compounds act at an extracellular A2/Ra purinoceptor. The xanthines, 1, 3-diethyl-8-phenylxanthine, 8-phenyltheophylline and theophylline antagonized the inhibitory action of N-ethyl-carboxamideadenosine competitively. Theobromine was inactive. This supports the view that the inhibitory receptor is of the A/R type. Hexobendine and dipyridamole, reported to antagonize the uptake of adenosine, failed to modify the response of human lung fragments to adenosine. The P site agonist 2',5' dideoxyadenosine inhibited the release of histamine and SRS-A. This effect was not prevented by the inhibitors of uptake, hexobendine and dipyridamole, nor was it antagonized by 8-phenyltheophylline.

Inhibition of adenosine accumulation by a CNS benzodiazepine antagonist (Ro 15-1788) and a peripheral benzodiazepine receptor ligand (Ro 05-4864).

Although benzodiazepines can inhibit adenosine uptake into central neurones, this effect is not antagonized by behaviourally effective 'benzodiazepine antagonists' such as Ro 15-1788. We now report that Ro 15-1788 and the 'peripheral' benzodiazepine ligand Ro 05-4864 themselves inhibit adenosine accumulation by rat brain synaptosomes. The inhibition of adenosine accumulation may thus underlie those behavioural effects of benzodiazepines which are mimicked but not antagonized by Ro 15-1788.

[Effect of hexobendine and pentoxifylline on the viscosity of erythrocyte suspension]. / Wirkung von Hexobendin und Pentoxifyllin auf die Viskosität von Erythrozytensuspensionen.

The effect of hexobendine, pentoxifylline and of the combination Instenon, containing hexobendine, on the dynamic viscosity of blood-isotonic and hyperosmolal suspensions of human and rat erythrocytes was studied. Hexobendine caused a statistically significant reduction of viscosity beginning at 10(-4) M, under hyperosmolal conditions even at 10(-5) M. Instenon was somewhat less active than hexobendine alone at a concentration equivalent to its presence in Instenon. Pentoxifylline did not decrease the viscosity under the selected conditions. The effect of hexobendine is considered due to an increase of red cell flexibility, probably caused by an elevation of the ATP content or by an alteration of the membrane structure of the erythrocytes.

Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists.

Uptake of [3H]adenosine into rat cerebral cortex synaptosomes was studied. Hexobendine (10(-5) M) and the benzodiazepine agonists diazepam (10(-5) M) and flurazepam (10(-4) M) significantly inhibited this uptake, but only if the compounds were pre-incubated for 10 min in the case of the benzodiazepines. The benzodiazepine antagonists Ro15-1788 (10(-5) M) and CGS 8216 (10(-5) M) failed to reverse the action of benzodiazepine agonists or hexobendine on [3H]adenosine uptake. The results add weight to the view that inhibition of adenosine uptake processes by benzodiazepines do not contribute to their behavioural effects.

Use of hexobendine to examine whether the coronary vasodilator metabolite released from guinea-pig isolated hearts is adenosine.

The coronary vasodilatation that accompanies cardiac stimulation by catecholamines and histamine or exposure to hypoxia is thought to be due to the release of a coronary vasodilator metabolite. Adenosine has been proposed as a likely candidate and this study examines its possible involvement by use of hexobendine, an agent that enhances the effects of adenosine. The force and rate of contraction and coronary perfusion pressure of isolated guinea-pig hearts were recorded. Adenosine exerted dose-dependent negative chronotropic effects and coronary vasodilatation. Both responses were potentiated during perfusion with hexobendine. However, hexobendine did not potentiate the coronary vasodilator responses to isoprenaline, suggesting that adenosine was not involved. Isolated hearts were then perfused in series, the donor heart supplying perfusate to the recipient. Stimulation of donor hearts by isoprenaline or histamine resulted in coronary vasodilator activity appearing in the recipient, which had received the appropriate antagonists of beta-adrenoceptors of histamine H1- and H2-receptors. Exposure of donor hearts to hypoxia also caused vasodilator metabolite release. Infusion of hexobendine into the recipient heats potentiated the vasodilator metabolite release. Infusion of hexobendine into the recipient hearts potentiated the vasodilator responses to exogenous adenosine, however, the responses to vasodilator metabolite released by the three procedures was not affected. Furthermore, there were no negative chronotropic responses accompanying the vasodilator metabolite release as there were with adenosine in a dose that produced equivalent vasodilatation. These results provide evidence that the vasoactive metabolite released by catecholamines, histamine and hypoxia is not adenosine.

[Pharmacologic modification of the circulation in the brain]. / Pharmakologische Beeinflussung der Zirkulation im Gehirn.

Hemispheric and regional cerebral blood flow were measured in 448 patients before and after application of various drugs. Of the tested substances, only a few affected hCBF: ephedrine-xanthines decreased hCBF, and midodrine, proxazole, vincamine, hexobendine, extract of ginkgo biloba, dextran, dextran-sorbitol, carboanhydrase inhibitor, and ouabain incresaed hCBF. In the rCBF maps different reaction patterns of regional flow were observed which were statistically tested by regression analysis. Homogeneous responses were seen as diffuse increases or decreases of rCBF. Heterogeneous responses occurred when perfusion of pathologic and normal areas reacted differently. An increase in rCBF in well-perfused areas with a shunt of blood from poorly supplied areas (intracerebral steal) was observed only with central vasodilators during the first days after an attack. Inverse cerebral steal phenomena with improvement of perfusion in the focus and decrease of flow in the surrounding brain may be elicited by different mechanisms. The effectiveness of drugs on cerebral circulation can be tested by measuring rCBF, but their therapeutic value for the treatment of patients with cerebrovascular disease must be shown in controlled clinical trials.

[Effects of hexobendine on peripheral circulation (author's transl)]. / Zur Wirkung von Hexobendin auf die periphere Zirkulation.

Hexobendine (Ustimon) given i.v. increases the arterial blood flow in the musculus tibialis anterior ninefold and improves the peripheral circulation for a limited time. Also the better arterial perfusion caused by adenosine and similar substances is intensified by hexobendine given i.v. These results are discussed with the already known effects of other coronary vasodilators.

The influence of Andiamina (hexobendine) on the histochemical reaction in the aorta wall of experimental animals.

The research was carried out on albino rats. The animals in the experimental group were given Andiamina (Hexobendine) in a dose of 40 mg/kg for a period of 7 days in the group I and 21 days in the group II. The results have pointed out that changes in the activity of the studied enzymes occurred especially after 21 days of Hexobendine administration. First of all, it caused a decrease in lactic and glucose-6-phosphate dehydrogenase activities and to lesser degree, it influenced the activities of iso-citric dehydrogenase and NAD and NADP tetrazole reductases. At the same time, reaction to succinic dehydrogenase indicated an increase in the enzymatic activity.

[Effects of hexobendine on adenosine metabolism and myocardial energy metabolism (author's transl)].

To elucidate the mode of action of hexobendine, its effects on some enzyme activities, the uptake of adenosine by rat erythrocytes and changes in the concentration of various myocardial substrates following induced hypoxia in rat were studied. Hexobendine had no effect on the in vitro activities of the adenosine degrading enzyme, adenosine deaminase and of the A-PRTase, HG-PRTase which are associated with the salvage pathways of purine biosyntheses. The uptake of adenosine by rat erythrocytes in vitro was inhibited considerably by hexobendine. Hypoxic states results in a significant decrease in creatine phosphate, ATP, glycogen and glucose contents, and increase in ADP, AMP, adenosine and lactate contents in rat myocardials. These alterations in cardiac metabolism induced by hypoxia were significantly improved by hexobendine given orally in doses of 10 approximately 100 mg/kg. Thus, hexobendine was shown to maintain the normal aerobic energy metabolism of the heart under states of hypoxia. In such states adenosine may be released from tissues and this increase in the available concentration of adenosine in plasma through inhibition of uptake by erythrocytes may be involved in the coronary vasodilating action of hexobendine.