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.

Effects of some purine derivatives on the guinea-pig trachea and their interaction with drugs that block adenosine uptake.

1 Adenosine, adenosine 5'-triphosphate (ATP), adenine, inosine and guanosine all caused concentration-dependent relaxations of guinea-pig tracheal smooth muscle in vitro. The relative potencies in descending order were: adenine greater than or equal to guanosine greater than inosine greater than or equal to adenosine greater than or equal to ATP. 2 Responses to the purine compounds were unaffected by propranolol (1 mug/ml). 3 The spasmolytic potencies of adenosine and ATP were greatly enhanced in the presence of the adenosine uptake blocking drugs dipyridamole, hexobendine or Dilazep, whereas responses to adenine were unaffected and those to inosine and guanosine were reduced. 4 The spasmolytic potencies of noradrenaline, aminophylline, prostaglandin E2 and glyceryl trinitrate were unaffected by dipyridamole, hexobendine and Dilazep. 5 It is suggested that an adenosine uptake process may exist in the trachea of the guinea-pig and that this process is inhibited by dipyridamole, hexobendine and Dilazep.

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 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.

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.

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.

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.

Differentiation of intestinal smooth muscle relaxation caused by drugs that inhibit phosphodiesterase.

In this study a number of chemically unrelated smooth muscle relaxants were tested: a) for potency of phosphodiesterase (PDE)-inhibition, using guinea pig colon-PDE and rat erythrocyte-PDE, b) for potency and duration of relaxation of the isolated guinea-pig colon, c) for their interaction with NH4Cl and orciprenaline as well as with "high calcium". Compared with the spasmolytic effect inhibition of guinea pig colon-PDE or rat erythrocyte-PDE was strong for papaverine and some other relaxants but was low or virtually absent with verapamil, hexobendine and bencyclane. The spasmolytic effect of some PDE-inhibitors was found diminished by the PDE-activator NH4Cl. Most of these drugs enhanced the relaxant effect of the "cyclase activator" orciprenaline; the latter are designated A-type drugs. Verapamil, bencyclane, hexobendine and M 13 did not show this type of interaction with NH4Cl and orciprenaline; they are designated B-type drugs. With A-type drugs--but not with B-type drugs--a highly significant correlation was observed between potency of relaxation and inhibition of colon-PDE (r = 0.85) as well as rat erythrocyte-PDE (r = 0.77). The spasmolytic action of aminophylline and papaverine (A-type drugs) was not inhibited by elevation of extracellular calcium to 9 mM, whereas relaxation induced by verapamil and hexobendine (B-type drugs) at 1.8 mM calcium was found abolished by 9 mM calcium. It is concluded that A-type drugs relax guinea-pig colon by inhibition of PDE and accumulation of cAMP, and that B-type drugs in all probability act by (a) cAMP-independent mechanism(s).

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.

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.

[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.

[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.

[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.

[Narcosis, platelet aggregation and arousing drugs]. / Narcosi, aggregazione delle piastrine e risveglianti.

During pentobarbital (25 mg/kg i.v. in 2 min), pentothal (15 mg/kg i.v. in 2 min) and ketamine (10 mg/kg i.v. in 2 min) narcosis, rabbits showed reduced platelet reactivity ot the direct aggregating effect of ADP (2 X 10(-5) M) and the indirect effect of thrombin (0.1 U/ml). Certain arousal drugs, specifically those of metabolic type such as SAMe (20 mg/kg i.v. in 2 min) and Thiola (166 mg/kg i.v. in 2 min) and of haemodynamic type such as nicergoline (6.66 mg/kg i.v. in 2 min) and hexobendine (5 mg/kg i.v. in 2 min) administered 31 min after narcosis induction, impede the depression brought on by narcosis on on platelet reactivity.

[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.

Influence of hexobendine, dipyridamole, dilazep, lidoflazine, inosine and purine riboside on adenosine uptake by the isolated epithelium of guinea pig jejunum.

The uptake of (14C)adenosine by isolated epithelium of guinea pig jejunum, administered on the blood side, was inhibited by hexobendine, dipyridamole, dilazep and lidoflazine. On the lumen side, however, weak inhibition was observed with lidoflazine only and no significant change was recorded with hexobendine, dipyridamole or dilazep. This difference was not altered when the degradation of hexobendine by the jejunal epithelium was blocked by physostigmine. When adenosine uptake was already reduced by purine riboside, further addition of hexobendine, dipyridamole, dilazep or lidoflazine caused divergent changes depending on the side of administration. Adenosine uptake was further diminished on the blood side, but raised towards control values on the lumen side. By contrast, inosine inhibited adenosine uptake on both sides of the epithleium. The results suggest that the mechanism of adenosine uptake is different on either side with respect to inhibition characteristics, corresponding to differences in morphology and function of the two sides of the intestinal epithelium.

Adenosine uptake by the isolated epithelium of guine pig jejunum.

The uptake of [8-14C]adenosine by the isolated epithelium of guinea pig jejunum was faster than that of inosine, hypoxanthine, or adenine. The initial velocity of adenosine uptake from both the luminal and the antiluminal side of the epithelium exhibited saturation kinetics. The apparent Km, V, and passive permeability of luminal adenosine uptake were all lower than the corresponding values of antiluminal uptake. p-Nitrobenzyl-thioguanosine inhibited adenosine uptake from both the luminal and the antiluminal side, whilst hexobendine decreased the uptake only from the antiluminal side of the epithelium. The results suggest that adenosine enters the intestinal epithelium by a carrier-mediated process in addition to passive diffusion. The antiluminal transport system for adenosine seems similar to that of other tissues with respect to hexobendine inhibition; the luminal transport mechanism, however, exhibits different properties, being insensitive to hexobendine.