Interaction of N-benzoyl-D-phenylalanine and related compounds with the sulphonylurea receptor of beta-cells.

1. The structure activity relationships for the insulin secretagogues N-benzoyl-D-phenylalanine (NBDP) and related compounds were examined at the sulphonylurea receptor level by use of cultured HIT-T15 and mouse pancreatic beta-cells. The affinities of these compounds for the sulphonylurea receptor were compared with their potencies for K(ATP)-channel inhibition. In addition, the effects of cytosolic nucleotides on K(ATP)-channel inhibition by NBDP were investigated. 2. NBDP displayed a dissociation constant for binding to the sulphonylurea receptor (K(D) value) of 11 microM and half-maximally effective concentrations of K(ATP)-channel inhibition (EC50 values) between 2 and 4 microM (in the absence of cytosolic nucleotides or presence of 0.1 mM GDP or 1 mM ADP). 3. In the absence of cytosolic nucleotides or presence of GDP (0.1 mM) maximally effective concentrations of NBDP (0.1-1 mM) reduced K(ATP)-channel activity to 47% and 44% of control, respectively. In the presence of ADP (1 mM), K(ATP)-channel activity was completely suppressed by 0.1 mM NBDP. 4. The L-isomer of N-benzoyl-phenylalanine displayed a 20 fold lower affinity and an 80 fold lower potency than the D-isomer. 5. Introduction of a p-nitro substituent in the D-phenylalanine moiety of NBDP did not decrease lipophilicity but lowered affinity and potency by more than 30 fold. 6. Introduction of a p-amino substituent in the D-phenylalanine moiety of NBDP (N-benzoyl-p-amino-D-phenylalanine, NBADP) reduced lipophilicity and lowered affinity and potency by about 10 fold. This loss of affinity and potency was compensated for by formation of the phenylpropionic acid derivative of NBADP. A similar difference in affinity was observed for the sulphonylurea carbutamide and its phenylpropionic acid derivative. 7. Replacing the benzene ring in the D-phenylalanine moiety of NBDP by a cyclohexyl ring increased lipophilicity, and the K(D) and EC50 values were slightly lower than for NBDP. Exchange of both benzene rings in NBDP by cyclohexyl rings further increased lipophilicity without altering affinity and potency. 8. This study shows that N-acylphenylalanines interact with the sulphonylurea receptor of pancreatic beta-cells in a stereospecific manner. Their potency depends on lipophilic but not aromatic properties of their benzene rings. As observed for sulphonylureas, interaction of N-acylphenylalanines with the sulphonylurea receptor does not induce complete inhibition of K(ATP)-channel activity in the absence of inhibitory cytosolic nucleotides.

Characterization of acetohexamide reductases purified from rabbit liver, kidney, and heart: structural requirements for substrates and inhibitors.

The structural requirements of acetohexamide reductases purified from rabbit liver, kidney, and heart for substrates and inhibitors were examined. Acetohexamide, an oral antidiabetic drug with a ketone group, and analogs of it with various alkyl groups instead of the cyclohexyl group were used as substrates for these three enzymes. The results obtained as to substrate specificity suggested that the nature of the substrate-binding region of the heart enzyme is markedly different from those of the substrate-binding regions of the liver and kidney enzymes. Tolbutamide, which has no ketone group within its chemical structure, strongly inhibited the heart enzyme, whereas it had little ability to inhibit the liver or kidney enzyme. The inhibition of the heart enzyme by tolbutamide was competitive with respect to acetohexamide and uncompetitive with respect to NADPH. Furthermore, tolbutamide analogs with n-pentyl and n-hexyl groups instead of the n-butyl group exhibited very pronounced inhibition of only the heart enzyme. Therefore, it is reasonable to postulate that the heart enzyme, unlike the liver and kidney ones, has a cleft of a strongly hydrophobic nature near its substrate-binding region, and that this hydrophobic cleft plays a critical role in the interaction of the heart enzyme with the cyclohexyl group of acetohexamide.

Divergent cardiac effects of the first and second generation hypoglycemic sulfonylurea compounds.

The effects of first and second generation hypoglycemic sulfonylureas on the incidence of ventricular ectopic beats and on the duration of transitional ventricular fibrillation in the ischemic rat heart were investigated. First generation sulfonylurea compounds (tolbutamide, carbutamide and gliclazide) in 105 preparations increased, while second generation sulfonylurea compounds (glibenclamide and glipizide) in 50 preparations decreased in a dose-dependent manner both the number of ventricular ectopic beats and the duration of transitional ventricular fibrillation during the first 30 min after ligation of the left anterior descending coronary artery. Therefore, second generation sulfonylureas should be preferred in the treatment of type 2 diabetics with ischemic heart diseases, if satisfactory metabolic control cannot be achieved by a treatment regimen and diet alone.

Electrophysiological effects of hypoglycaemic sulphonylureas on rabbit heart.

The hypoglycaemic sulphonylurea drugs glibenclamide and carbutamide were compared as to their influence on the electrical activity of isolated rabbit heart preparations. Carbutamide (10(-5)-10(-3) M) was found to depress slightly the nomotopic and junctional automaticity, while in Purkinje fibres it enhanced automaticity and conduction velocity. On the other hand, glibenclamide (10(-6)-10(-3) M) had no significant influence on the nomotopic and junctional automaticity, and markedly depressed automaticity and conduction velocity in the Purkinje fibres. The atrial conduction system was not much influenced by the sulphonylureas tested. Carbutamide depressed the electrical threshold in Purkinje fibres. It follows that the hypoglycaemic sulphonylurea compounds carbutamide and glibenclamide differ considerably with respect to their influences on cardiac electrical activity.

[The effect of repeated applications of carbutamide on the embryonic development of the rat (author's transl)]. / Die Wirkung der mehrmaligen Gaben von Carbutamid auf die Keimlingsentwicklung der Ratte.

The present paper will check the effects of repeated applications of the oral "antidiabeticum" Carbutamide (substitute of the "sulfonylurea") on the embryonic development of wistar rats. The agent was given the pregnant animal by the means of a throat sonde at the 5th, 6th and 7th day post coitum suspended in Tween 80 in a dosage of 800 mg/kg bodyweight. In order to value the intrauterine development the following parameters were used: mean implantation rate, resorptions rate, bodyweight of fetuses, statemend of animalies by an accurate inspection of the body surface shape, of the skeleton and the inner organs. The rates of implantation do not differ from that of the control group. The rate of resorption increases dependend on the beginning of the treatment. The later the treatment begins the higher the resorptions rate. The body weight of surviving fetuses is diminished strongly. Damage of the skeleton appears as the presence of 14th ribs at one or each side or as ossifications at the 7th neck vertebra (neck rib). Also this results depend on the beginn of the treatment.

[Effect of insulin on germination and ionic exchange in Raphanus sativus (author's transl)]. / Efectos de la insulina sobre la germinación e intercambio iónico del Raphanus sativus.

Hypoglycemic sulfamide BZ-55 activates or inhibits germination of Raphanus sativus, depending upon the dosis. Since this drug acts upon the glycemia by increasing the secretion and action of insulin, the influence of this hormone on germination and ionic changes (Na+-K+) between seeds and culture medium, were studied. Seeds were incubated during 72 h with different concentrations of insulin in 10 ml deionized water or in 10 ml 18 mM K+ (KCl) solutions at 37 degrees C in vapor saturated atmosphere. A solution of 0.125 IU insulin/ml in water increases the germination to 110% whereas 0.175 IU insulin/ml inhibits it to 40% against controls. Further increases in insulin concentration always inhibit germination. Similar results have been obtained with K+ containing media. Germination rate changes in a small concentration range suggest that insulin might affect an enzymatic activity in the seed.

The direct effect of hypoglycaemic sulphonylureas on myocardial contractile force and arterial blood pressure.

The cardiovascular effects of glibenclamide, gliclazide and carbutamide were studied on myocardial contractile force, cardiac output, mean arterial pressure and heart rate in 43 intact, 14 pancreatectomized and 18 adrenalectomized dogs. It is shown that a positive inotropic and hypertensive effect of hypoglycaemic sulphonylureas develops without the mediation of glucagon, insulin or adrenaline. Contrary to the other investigated sulphonylureas, glibenclamide reduced myocardial contractile force and arterial blood pressure.

[Kinetics of sodium thiosulfate-35S fixation and effect of some sulfur compounds on iodine-131 fixation in ray thyroid gland]. / Cinétique de fixation thyroïdienne du thiosulfate de sodium-35 S et effets de quelques molécules soufrées sur la fixation thyroïdienne de l'iode 131 chez le rat.

In order to invalidate or confirm the affirmation that non antithyroid sulphide molecules alter the measure of the thyroid fixation rate of iodine 131 we undertook on the rat: on one hand a kinetic study of thyroid fixation of sodium thiosulfate labelled with sulphur 35, which showed a very low captation not exceeding 0,01% of injected radioactivity; on the other hand the study of the effects of some sulphide molecules on thyroid fixation of iodine 131 in the rat: sodium thiosulfate, association of sodium thiosulfate + metalloidal sulphur + methionine, carbutamide and dimethylsulfoxyde in various kinds of dose administration and periods. None of the products used in our work conditions produced a significant decrease of the fixation rate of iodine 131. In three different experimental protocols (sulfur association and dimethylsulfoxide), we showed a significant light increase of the fixation rate.

Comparison of the effects of leucines, non-metabolizable leucine analogues and other insulin secretagogues on the activity of glutamate dehydrogenase.

Glutamate dehydrogenase (GLDH) from bovine liver was employed in model system for testing a possible role of GLDH in insulin release. The ability of different insulin secretagogues to stimulate the activity of the diethylstilbestrol-inhibited enzyme was tested. The two insulin-releasing amino acids, L-leucine and its non-metabolizable analogue 2-aminobicyclo(2, 2, 1)heptane-2-carboxylic acid [b(--)-BCH], were the best stimulators of GLDH activity. The non-secreting stereoisomers, D-leucine and b(+)-BCH, were less effective. Glucose, L-arginine and the leucine metabolite alpha-ketoisocaproic acid lacked significant effects on GLDH activity. Small and diverging effects were obtained with sulfonvlurea compounds: whereas carbutamide caused slight stimulation, tolbutamide and glipizide had no effect, and glibenclamide was an inhibitor. The specificity of the insulin-releasing amino acids L-leucine and b(--)-BCH in stimulating GLDH activity makes it tempting to speculate about a connection between allosteric regulation of pyridine nucleotide-dependent enzymes and insulin release.