Ingelore Hackbarth

Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber

1 The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2 In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy‐demethyl tacrolimus were generated. Their formation was concentration‐ and time‐dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3 The metabolite formation was highest in the duodenum and declined in the order duodenum > 4 Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5 Tacrolimus metabolites re‐entered the mucosa chamber (>90%) and passed through the small intestinal preparation into the serosa chamber. 6 It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re‐enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus

Mechanism of gastric antisecretory effect of SCH 28080

1 The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea‐pig and guinea‐pig gastric membranes purified and enriched with K+/H+‐ATPase. 2 In guinea‐pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. 3 SCH 28080 inhibited the purified K+/H+‐ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 μM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+‐ATPase showed that this enzyme was only slightly depressed by SCH 28080. 4 It is concluded that SCH 28080 acts with high selectivity on the parietal cell K+/H+‐ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+‐site of the gastric ATPase.

DRUG INTERACTIONS AND INTERINDIVIDUAL VARIABILITY OF CICLOSPORIN METABOLISM IN THE SMALL INTESTINE

The undecapeptide ciclosporin is used as immunosuppressant after organ transplantation and for therapy of immune diseases. Low and variable bioavailability of ciclosporin has been attributed to its metabolism in the small intestine. The aim of the present study was to investigate drug interactions and interindividual variability of ciclosporin metabolism in the small intestine. Ciclosporin metabolism was studied in vitro using microsomes isolated from the small intestine of humans and pigs. The metabolites generated were quantified by HPLC and identified by mass spectrometry. Using specific antibodies and inhibitors, we showed that, as in the liver, cytochrome P450 3A (CYP 3A) enzymes are responsible for ciclosporin metabolism in the human small intestine. Of the 28 xenobiotics included in the study, 16 drugs, all well-known CYP 3A inhibitors, inhibited ciclosporin metabolism in the small intestine. In the small intestine of different patients, the rate of metabolism varied by a factor of 10. Ciclosporin was metabolized faster by small intestine microsomes from female (n = 4) than from male (n = 10) patients (p < 0.009).

The positive inotropic effect of phenylephrine in the presence of propranolol. Increase in time to peak force and in relaxation time without increase in c-AMP

SummaryThe effects of phenylephrine on the shape of the contraction curve and on the cyclic adenosine 3′,5′-monophosphate (c-AMP) content were studied in electrically driven (frequency 0.2 Hz) cat papillary muscles. All experiments were done in the presence of 1 μM propranolol in order to minimize interference from β-adrenoceptors. 1.Phenylephrine increased the force of contraction in a concentration-dependent manner. Maximal effects (about 200% of control) occurred at 30 μM phenylephrine.2.The positive inotropic effect (PIE) of phenylephrine was antagonized by phentolamine. Phentolamine, 5μM, produced a parallel shift of the concentration-response curve for the PIE of phenylephrine by about two log units to the right.3.The PIE of 30 μM phenylephrine occurred without any detectable increase in the c-AMP levels of the preparations.4.The PIE of 30μM phenylephrine developed about three times more slowly than the PIE of an equieffective concentration of isoprenaline.5.The PIE of phenylephrine was accompanied by significant, concentration-dependent increases in both time to peak force and relaxation time.6.It is concluded that the PIE of phenylephrine in the presence of propranolol is mediated mainly by a stimulation of α-adrenoceptors. It is unlikely to be related to an increase in c-AMP. With respect to time course and influence on the shape of the contraction curve it is qualitatively different from the effects of β-adrenoceptor stimulation. These data are taken to support the hypothesis that the mechanical effects of α-and β-adrenoceptor stimulating agents on the heart are produced by different mechanisms.

Effects of vanadate on the c-AMP system of the heart

Summary300 μM vanadate (NH4VO3) caused an about 65% increase in force of contraction in isolated electrically driven cat papillary muscles. The positive inotropic effect (PIE) was accompanied by only a small (about 20%) but significant increase in c-AMP levels. 0.3 μM isoprenaline (ISO) produced a much greater increase in c-AMP levels (about 80%) while the PIE of ISO was as great as the PIE observed with 300 μM NH4VO3. In cat right ventricular adenylate cyclase preparations NH4VO3 and ISO stimulated adenylate cyclase activity by nearly the same extent. Propranolol (1 μM and 5 μM) prevented the ISO-induced adenylate cyclase stimulation but not the stimulation due to NH4VO3. Phosphodiesterase activity was not affected by NH4VO3 (up to 1000 μM). It is concluded that the small vanadate-induced increase in c-AMP levels, which is due to adenylate cyclase stimulation, inintact papillary muscles maycontribute to its PIE, but other mechanisms may be at least equally important.Zusammenfassung300 μM Vanadat (NH4VO3) steigerte an isolierten, elektrisch gereizten Katzenpapillarmuskeln die Kontraktionskraft um ca. 65%. Der positiv inotrope Effekt (PIE) ging einher mit einem nur geringen (ca. 20%), aber signifikanten Anstieg des c-AMP-Gehaltes. 0.3 μM Isoprenalin (ISO) verursachte einen sehr viel größeren Anstieg des myokardialen c-AMP-Gehaltes (ca. 80%), während der PIE von ISO gleich groß war wie der PIE von NH4VO3. In einer Adenylatcyclasepräparation von rechten Ventrikeln des Katzenherzens stimulierten NH4VO3 und ISO die Adenylatcyclaseaktivität in nahezu gleichem Ausmaß. Propranolol (1 μM und 5 μM) verhinderte die Aktivierung der Adenylatcyclase durch ISO, aber nicht die Aktivierung durch NH4VO3. Die Phosphodiesteraseaktivität wurde durch NH4VO3 (bis zu einer Konzentration von 1000 μM) nicht beeinflußt. Aus den Ergebnissen wird geschlossen, daß der geringe vanadatinduzierte c-AMP-Anstieg, der über eine Stimulation der Adenylatcyclase zustande kommt, zwar am Zustandekommen des PIE beteiligt sein mag, daß aber andere Mechanismen von mindestens ebenso großer physiologischer Bedeutung sind.

Effect of Substituted Benzimidazoles on H+K+ATPase of Isolated Guinea-pig Parietal Cells

Omeprazole, a substituted benzimidazole, has been shown to inhibit gastric acid secretion in vitro and in vivo. In vitro, omeprazole inhibits acid secretion stimulated at all cellular levels, including that stimulated by K+, indicating that it is an inhibitor of the parietal cell proton pump. The present paper describes the inhibitory effect of substituted benzimidazoles, such as picoprazole and omeprazole, on the H+K+ATPase isolated from guinea-pig parietal cells, and the reversibility of acid inhibition in isolated guinea-pig parietal cells by the thiol compound, dithiothreitol.

The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa

In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H+ secretion, as measured by 14C-aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10(6) cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H+ secretion, but that the acid response elicited by histamine requires another intracellular pathway.

Effect of vanadate on myocardial force of contraction

SummaryAmmonium vanadate (NH4VO3; 50–1000 μM) increased the force of contraction of isolated electrically driven cat papillary muscles in a concentration-dependent manner. The positive inotropic effect (PIE) of NH4VO3 became significant at 50 μM and was maximal at 500 to 1000 μM. It was accompanied by an increase in the rate of force development, in the rate of relaxation and in relaxation time of the isometric contraction. Similar results as with NH4VO3 were obtained with NaVO3 and with Na3VO4. The effects of NH4VO3 were also observed in the presence of 1 μM propranolol, 5 μM phentolamine or after reserpine-pretreatment (5 mg/kg i.p.). These results indicate that vanadate produces a direct PIE inventricular cardiac muscle which is unlikely to be mediated by alpha-or beta-adrenoceptor stimulation. In cat left atrial strips, however, vanadate ions produced a negative inotropic effect through a hitherto unknown mechanism. Vanadate effects similar to those observed in the cat heart were obtained in ventricular and atrial preparatins from bovine hearts.ZusammenfassungDie Wirkung von Vanadat (NH4VO3, NaVO3, Na3VO4) auf die Kontraktionskraft des Herzens wurde an isolierten, elektrisch gereizten Katzenpapillarmuskeln untersucht. NH4VO3 (50–1000 μM) wirkte konzentrationsabhängig positiv inotrop. Der Effekt begann nach etwa 1 min, war nach 6–10 min maximal und blieb während der gesamten Versuchsdauer (30 min) erhalten. Die Wirkung von Vanadat wurde auch in Gegenwart von Propranolol und Phentolamin oder nach Reserpinvorbehandlung beobachtet. NH4VO3 steigerte Anstiegs- und Erschlaffungsgeschwindigkeit der isometrischen Kontraktion. Die Kontraktionsdauer wurde verlängert; dieser Effekt beruhte auf einer Verlängerung der Erschlaffungszeit. Mit NaVO3 und Na3VO4 wurden ähnliche Ergebnisse erzielt. Die Versuche sprechen dafür, daß Vanadat anventrikulären Herzmuskelpräparaten direkt positiv inotrop wirkt und daß diese Wirkung nicht durch Alpha- oder Beta-Adrenozeptoren vermittelt wird. An linken Katzenvorhofstreifen führte Vanadat zu einem negativ inotropen Effekt, dessen Ursache bisher ungeklärt ist. An isolierten Ventrikel- und Vorhofstrabekeln aus Rinderherzen hatte Vanadat ähnliche Effekte wie am Katzenherzen.