Novel inhibitors of the sodium-calcium exchanger: benzene ring analogues of N-guanidino substituted amiloride derivatives.

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium-calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called 'simplified analogues', where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium-calcium exchanger was screened on both cell types by measuring their effect on 45Ca(2+) uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N'-(1-naphtylmethyl)guanidine (IC(50)=3.4 microM) was found more active than amiloride (IC(50)=690 microM) and 3,4-dichlorobenzamil (IC(50)=15.2 microM), the reference inhibitor.

3-and 4-substituted 4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides as potassium channel openers: synthesis, pharmacological evaluation, and structure-activity relationships.

4-N-Substituted and -unsubstituted 3-alkyl- and 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides were synthesized and tested vs diazoxide and selected 3-alykl- and 3-(alkylamino)-7-chloro-4H-1,2,4-benzothiadiazine 1,1-dioxides as potassium channel openers on pancreatic and vascular tissues. Several 4-N-unsubstituted 3-(alkylamino)pyridothiadiazines and some 3-(alkylamino)-7-chlorobenzothiadiazines were found to be more potent than diazoxide for the inhibition of the insulin-releasing process. Moreover, the 3-(alkylamino)pyridothiadiazines appeared to be more selective for the pancreatic than for the vascular tissue. By means of the pharmacological results obtained on pancreatic B-cells, structure--activity relationships were deduced and a pharmacophoric model for the interaction of these drugs with their receptor site associated to the pancreatic K(ATP) channel was proposed. According to their selectivity for the B-cell (endocrine tissue) vs the vascular (smooth muscle tissue) ionic channel, selected 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4,-thiadiazine 1,1-dioxides may serve as pharmacological tools in studying the K(ATP) channels ("pancreatic-like" K(ATP) channels) in other tissues.

Hexose metabolism in pancreatic islets: glycogen synthase and glycogen phosphorylase activities.

The activity of glycogen synthase and glycogen phosphorylase was measured in rat pancreatic islet homogenates. For this purpose, the sensitivity of current radioisotopic procedures for the assay of these enzymes in liver extracts was increased by about two orders of magnitude. Even so, the measurement of glycogen synthase and phosphorylase in islet homogenates was hampered by a potent amylase-like activity, resulting in the hydrolysis of preformed or newly formed 14C-labeled glycogen. Acarbose suppressed the latter phenomenon which was found attributable to both minute contamination of isolated islets by acinar cells and genuine alpha-amylase activity in purified islet beta-cells. As measured by the more sensitive method in the presence of acarbose, the a/(a+b) ratio for glycogen synthase activity in islet homogenates was increased in islets preincubated in the presence as distinct from absence of D-glucose and decreased after preincubation with forskolin. These changes represented a mirror image of those evoked by D-glucose and forskolin in the a/(a+b) ratio for glycogen phosphorylase activity. It is concluded that glycogen synthesis and breakdown are regulated in the endocrine pancreas in a manner qualitatively comparable to that prevailing in hepatocytes, the possible participation of an amylase-like activity to glycogen metabolism in intact islet beta-cells requiring further investigation.

Is glycolysis impaired in erythrocytes of diabetic rats?

In erythrocytes from diabetic rats the generation of radioactive metabolites from 3H- or 14C-labeled D-glucose occurs at a lower rate than in cells from control animals, while the total production of L-lactic acid is the same in both cell types. The radioisotopic anomaly is not corrected by agents causing phosphofructokinase activation and is not attributable to interference of metabolites generated from either glycogen or 2,3-diphospho-D-glycerate. It is mimicked by preincubating the erythrocytes at a high concentration of D-glucose and suppressed in diabetic rats treated with insulin in order to lower the blood glucose concentration prior to sacrifice. These findings suggest isotopic dilution of the 3H- or 14C-labeled tracer by endogenous unlabeled D-glucose. The influence of factors such as the concentration of erythrocytes in the incubation medium, the concentration of extracellular D-glucose, and the length of incubation upon radioactive metabolic variables is also compatible with the postulated phenomenon of isotopic dilution.

Interference of glycogenolysis with glycolysis in pancreatic islets from glucose-infused rats.

When pancreatic islets isolated from rats infused for 48-72 h with a hypertonic solution of D-glucose were incubated for two successive periods of 10 min each, in the presence first of 16.7 mM and then 2.8 mM D-[U-14C]glucose, the total output of L-lactic acid during the second incubation was as high as that recorded during the first incubation, while the specific radioactivity of L-lactic acid dramatically decreased during the second incubation. In islets from normoglycemic rats, however, the total output of L-lactic acid decreased and its specific radioactivity modestly increased as the concentration of D-glucose was lowered from 16.7 to 2.8 mM. Such contrasting results indicate that in the glycogen-rich islets isolated from glucose-infused rats, the fall in extracellular D-glucose concentration was not accompanied by a parallel fall in glycolytic flux, the decreased utilization of exogenous D-[U-14C]glucose coinciding with stimulation of glycogenolysis. This unusual metabolic situation also coincided with a transient and paradoxical stimulation of insulin release in response to the decrease in extracellular D-glucose concentration. It is proposed, therefore, that the interference of glycogenolysis with glycolysis in pancreatic islets from glucose-infused rats participates in the paradoxical changes in insulin output which represent a typical feature of B-cell glucotoxicity.

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes.

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.