Inhibition of the ATP-sensitive potassium channel by class I antiarrhythmic agent, cibenzoline, in rat pancreatic beta-cells.

1. Cibenzoline, a class I antiarrhythmic agent, was investigated for its effect on the ATP-sensitive K+ channel of pancreatic beta-cells by the patch clamp technique. 2. In perforated patch clamp experiments, cibenzoline depolarized the membrane of single beta-cells and thereafter, caused firing of action potentials in the presence of 2.8 mM glucose. 3. Cibenzoline inhibited the activity of the ATP-sensitive K+ channel in cell-attached recordings in the presence of 2.8 mM glucose and evoked repetitive fluctuations of the baseline current, apparently reflecting the action potentials of the beta-cell. 4. In whole-cell clamp experiments, time-independent outward current was induced by depleting cytoplasmic ATP with 0.1 mM ATP and 0.1 mM ADP in the solution contained in the pipette. The outward current was inhibited by cibenzoline in a dose-dependent manner in the concentration range of 1 microM to 100 microM and half maximum inhibition occurred at 1.5 microM. 5. Cibenzoline blocked substantially the ATP-sensitive K+ channel current when applied at the inner side of the membrane in isolated inside-out membrane patches. 6. It is concluded that cibenzoline blocks the ATP-sensitive K+ channel of pancreatic beta-cells and, thereby, stimulates insulin secretion at sub-stimulatory levels of glucose.

Stimulation by volume expansion of atrial natriuretic peptide release from perfused rat heart.

The release of immunoreactive (ir-) rat atrial natriuretic peptide (rANP) with volume expansion in in situ retrograde perfused rat heart was examined. The volume expansion induced by the infusion of the perfusion medium into the right atrium increased the mean right atrial pressure and the ir-rANP release without changing the rate of the heart beat. There was a significant correlation between the peak values of ir-rANP release and those of mean atrial pressure. The bilateral cervical vagotomy did not effect the ir-rANP release induced by the volume expansion. Therefore, it is highly likely that the stimulatory effect of volume expansion on rANP release is due to, at least in part, the atrial distension accompanied by an increase in mean atrial pressure, not involving a vagal system.

Regional differences in calcium sensitivity in the guinea-pig intestine.

The effect of the Ca(2+)-channel antagonist nicardipine on the basal tone of six segments (duodenum, jejunum, ileum, proximal colon, distal colon and rectum) of the guinea-pig intestine has been investigated in muscle preparations. Nicardipine reduced the basal tone of the proximal and distal colon but not of the duodenum, jejunum, ileum and rectum. Similarly, when each segment was incubated in Ca(2+)-free medium, the basal tone of the proximal and distal colon, but not that of the other four segments, was reduced. The reduced basal tone recovered after cumulative addition of Ca2+ in both colon preparations. The basal tone of the distal colon was partly reduced by tetrodotoxin, atropine and clonidine. Conversely, L-type Ca(2+)-channel antagonists (Cd2+, verapamil and nicardipine), but not the T-type Ca(2+)-channel antagonist Ni2+, completely reduced the basal tone of the distal colon. These results indicate that in the regulation of basal tone there are additional regional differences in the effect of Ca2+ influx into the cells from the extracellular fluid which might involve L-type-like Ca2+ channels and might partly be because of neuronal factors.

Anti-gastric acid secretory mechanism of 1,6-dihydro-2-[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidinecarboxylic acid. Effect on mucosal mast cell.

To clarify the anti-gastric acid secretory mechanism of 1,6-dihydro-2-[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidinecarbo xylic acid (CAS 98772-05-5, MAR-99), the relationship between gastric acid secretion and gastric mucosal mast cell (MMC) was studied and the effect of this compound on these parameters was examined and compared with anti-allergic drugs (mast cell stabilizers) and anti-ulcer drugs. The release of histamine from MMC cultured from bone marrow and connective tissue mast cell (CTMC) isolated from peritoneal cavity was found to be induced by the addition of ethanol (final conc. 17.5%), and the inhibitory effect on histamine release from MMC is closely associated with the anti-gastric secretory effect. That is to say, MAR-99 (10(-9)-10(-7) mol/l) inhibited histamine release from MMC induced by ethanol in a concentration-dependent manner. The action of MAR-99 on MMC was more sensitive than that of CTMC. In addition, MAR-99 (100 mg/kg i.d.) suppressed gastric acid secretion. On the other hand, anti-allergic drugs (mast cell stabilizers), such as DSCG and tranilast (both 10(-7) mol/l), markedly inhibited histamine release from CTMC induced by ethanol, but these drugs (10(-8)-10(-7) mol/l) showed only a tendency to prevent the release of histamine from MMC. Furthermore these drugs (both 100 mg/ kg i.d.) had no effects on gastric acid secretion. Equally anti-ulcer drugs, such as cetraxate, teprenone and sofalcone, had no effects on histamine release from mast cells of two types and gastric acid secretion. From these results, it was suggested that MMC is closely correlated with gastric acid secretion, and the anti-gastric secretory effect of MAR-99 may mainly contribute to prevent the degranulation of MMC.

Modulation of IgE synthesis by IgE-binding factors released by T cells of asthmatic patients with elevated serum IgE.

The culture supernatants of unstimulated T cells (TCS) from asthmatic patients with elevated serum IgE were tested for IgE-binding factors (IgE-BFs) displaying the IgE-potentiating activity. The IgE-BFs were detected by their ability to inhibit the rosetting of RPMI 8866 cells with ox erythrocytes coupled with mouse monoclonal antibody (E-Mab) specific to Fc receptors for IgE (Fc epsilon R). TCS showing the rosette-inhibiting activity significantly enhanced the spontaneous IgE synthesis by B cells of allergic individuals. Interestingly, rosette-inhibiting factors could be removed by absorption with IgE-Sepharose from which they were subsequently eluated with acid buffer, indicating that the rosette inhibition was indeed mediated by IgE-BFs. In addition, such IgE-BFs had affinity for concanavalin A and lost their IgE-potentiating activity after treatment with trypsin and neuraminidase. In contrast, T cells treated with tunicamycin released IgE-suppressing factors capable of inhibiting the IgE-potentiating activity of TCS derived from untreated T cells. On the other hand, the culture supernatants from subpopulations depleted of Fc epsilon R+ T cells but not of Fc gamma R+ T cells contained neither rosette-inhibiting factors nor IgE-potentiating factors, suggesting that IgE-BFs were released by in vivo pre-activated Fc epsilon R+ T cells. With regard to circulating Fc epsilon R+ T cells determined by E-Mab, they were significantly higher in asthmatic patients with elevated serum IgE (0.77 +/- 0.15%) than in normal subjects (0.17 +/- 0.07%) in spite of a very small proportion of T cells bearing Fc epsilon R.

The effects of intraperitoneal injection of 6-hydroxydopamine on the turnover and the levels of the brain catecholamines and the levels of plasma corticosterone in rats.

1. The effects of intraperitoneal injection of 6-hydroxydopamine (6-OHDA) on the levels and the turnover of brain catecholamines and the levels of plasma corticosterone were studied in rats. 2. Two weeks after intraperitoneal injection of 6-OHDA (150 mg/kg) a virtually complete disappearance of cardiac noradrenaline was observed. 3. An increment and an accelerated turnover of noradrenaline in the hypothalamus was observed 2 weeks after peripheral administration of 6-OHDA (150 mg/kg). 4. There was no change in the levels and the turnover of noradrenaline in the cortex of the rats so treated. 5. There was not change in the levels and the turnover of dopamine in either the hypothalamus or the cortex of the 6-OHDA-treated rats. 6. An increment and an accelerated turnover of hypothalamic noradrenaline were not associated with any change in plasma corticosterone.

Effect of 1,6-dihydro-2[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidineca rboxyli c acid on ethanol-induced microvascular injury in rats.

To disclose the cytoprotective mechanism of 1,6-dihydro-2[2-(2-methyoxypropoxy)anilino]-6-oxo-5-pyrimidinecarb oxylic acid (CAS 98772-05-5, MAR-99), the effect of this compound on the microvascular injury in gastric mucosa induced by 99.5% ethanol in rats was studied. In this experiment, it was found that the elevation of vascular permeability observed at the early state of ethanol-induced gastric mucosal injury was closely correlated with the combined action of histamine and slow reacting substance (leukotriene C4, LTC4). MAR-99 (0.3-10 mg/kg p.o.) prevented dose-dependently the increase in vascular permeability. Furthermore, MAR-99 (10 mg/kg p.o.) improved the decrease in the number of histamine containing cells and histamine content, and prevented the production of LTC4. These results suggest that MAR-99 exerts its anti-microvascular injury effect by regulating the release of histamine and the production of LTC4 in glandular stomach against ethanol, and this effect may contribute to the anti-lesion effect of this compound.

Increased sensitivity to 5-hydroxytryptamine due to intraventricular administration of 5,6-dihydroxytryptamine.

The possibility that intraventricular administration of 5,6-dihydroxytryptamine (5,6-DHT) can cause a development of denervation hypersensitivity to central 5-hydroxytryptamine (5-HT) was examined in rats with special reference to the pituitary-adrenocortical functions. Pretreatment with intraventricular injection of 5,6-DHT itself did not affect the basal concentrations or stress-induced increases of plasma B. The same pre-treatment, however, elicited augmented responses to subcutaneous injection of 5-HTP, i.e. there was a significant rise in the concentrations of plasma B as compared with those in the controls. These results suggest a development of denervation hypersensitivity to 5-HT following intraventricular injection of 5,6-DHT.

Anti-ulcer effect of 1,6-dihydro-2-[2-(2-methylpropoxy) anilino]-6-oxo-5-pyrimidinecarboxylic acid on experimental gastric ulcers in rats.

The effects of 1,6-dihydro-2-[2-(2-methylpropoxy) anilino]-6-oxo-5-pyrimidinecarboxylic acid (MAR-99, CAS 98772-05-5) on various experimental gastric ulcers were studied in rats. MAR-99 (3-100 mg/kg, p.o. or i.d.) showed the anti-ulcer effect in Shay-, stress, acetylsalicylic acid (ASA)- and compound 48/80-ulcer models and significantly accelerated healing of acetic acid-induced gastric ulcer in rats. In addition, MAR-99 (1-10 mg/kg p.o.) decreased dose-dependently the gastric mucosal damage induced by necrotizing agents such as 99.5% ethanol, 0.6N HCl and 0.2 N NaOH. These results indicate that MAR-99 may be useful for the treatment of gastric ulcer in human.

Studies on antiulcer agents. IV. Antiulcer effects of 2-benzylthio5,6,7,8-tetrahydro-4(3H)-quinazolinones and related compounds.

With a view to finding more effective antiulcer agents, a series of 2-benzylthio-5,6,7,8-tetrahydro-4(3H)-quinazolinones and related compounds were synthesized and evaluated in a histamine-stimulated gastric secretion model. The sodium salt of the 2-(dimethylamino)benzylthio derivative (8) showed gastric mucosal protection and gastric antisecretion activities, and was also effective against experimental gastric and duodenal ulcers induced by some ulcerogenic agents. Based on a comparison of the antiulcer properties of 8 with those of the lead compounds (1 and 2) and cimetidine, it appears that, for improvement of antiulcer activity, the reduction of gastric acidity is a more important factor than the reduction of gastric volume output or gastric total acid output.

Effect of 1,6-dihydro-2-[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidinecarboxylic acid on gastric mucosal defensive factors and gastric secretion in rats.

1,6-dihydro-2-[2-(2-methylpropoxy)anilino]-6-oxo-5-pyrimidinecarbo xylic acid, (MAR-99, CAS 98772-05-5) (10-30 mg/kg i.g.) improved the reduction of gastric blood flow rate induced by the administration of 99.5% ethanol or acidified-acetylsalicylic acid (ASA). In addition, MAR-99 (3 x 10(-6)-3 x 10(-5) mol/l) protected dose-dependently the damage of epithelial cells induced by ulcerogenic agents such as ethanol or acidified-ASA. MAR-99 (1-10 mg/kg p.o.) prevented dose-dependently the reduction of hexosamine content in glandular stomach. Furthermore, MAR-99 (10-30 mg/kg i.g.) improved the decrease in gastric potential difference induced by 99.5% ethanol and acidified-ASA. MAR-99 (10-30 mg/kg p.o.) significantly inhibited the lesion formation induced by 99.5% ethanol and such effect of this compound was not attenuated by the pretreatment with indomethacin. Furthermore MAR-99 (10 and 30 mg/kg p.o.) had no effect on the prostaglandins (PGE2 and I2) contents in the stomach of normal rats. In pylorus-ligated rats, MAR-99 (3-100 mg/kg i.d.) showed a weak or no effect on acidity and pepsin activity in gastric juice, although this compound decreased dose-dependently the volume of gastric juice. In perfused stomachs, MAR-99 (30-100 mg/kg i.d.) slightly prevented the acid secretion induced by carbachol and pentagastrin. However, MAR-99 did not affect the acid secretion stimulated by histamine. These results indicated that anti-ulcer effect of MAR-99 was mainly due to maintenance of the gastric mucosal resistance.

Studies on antiulcer agents. II. Antiulcer properties of N-(1H-tetrazol-5-yl)-2-anilino-5-pyrimidinecarboxamides inhibiting release of histamine from passively sensitized rat peritoneal mast cells.

With the aim of applying mast cell-stabilizing agents as antiulcer agents, N-(1H-tetrazol-5-yl)-2-anilino-5-pyrimidinecarboxamides were synthesized, and initially evaluated pharmacologically for activity in the rat passive cutaneous anaphylaxis test by oral administration. The most active compound 6 was proved to inhibit potently the release of histamine from passively sensitized rat peritoneal mast cells in vitro. When compared with other mast cell-stabilizing agents and an antiulcer agent, compound 6 was found to show excellent gastric mucosal protection and gastric antisecretion activities. Furthermore, compound 6 revealed good activity against acidified aspirin ulcer in rats and water-immersion stress ulcer in rats.