HET0016, a potent and selective inhibitor of 20-HETE synthesizing enzyme.

The present study examined the inhibitory effects of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016) on the renal metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes. HET0016 exhibited a high degree of selectivity in inhibiting the formation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) in rat renal microsomes. The IC(50) value averaged 35+/-4 nM, whereas the IC(50) value for inhibition of the formation of epoxyeicosatrienoic acids by HET0016 averaged 2800+/-300 nM. In human renal microsomes, HET0016 potently inhibited the formation of 20-HETE with an IC(50) value of 8.9+/-2.7 nM. Higher concentrations of HET0016 also inhibited the CYP2C9, CYP2D6 and CYP3A4-catalysed substrates oxidation with IC(50) values of 3300, 83,900 and 71,000 nM. The IC(50) value for HET0016 on cyclo-oxygenase activity was 2300 nM. These results indicate that HET0016 is a potent and selective inhibitor of CYP enzymes responsible for the formation of 20-HETE in man and rat.

N-linked oligosaccharide processing enzyme glucosidase II produces 1,5-anhydrofructose as a side product.

alpha-1,4-Glucan lyase cleaves alpha-1,4-linkages of nonreducing termini of alpha-1,4-glucans to produce 1,5-anhydrofructose (1,5-AnFru). The enzymes isolated from fungi and algae show high homology with glycoside hydrolase family 31. Purification of alpha-1,4-glucan lyase from rat liver using DEAE Cellulose chromatography resulted in separation of two enzymatic active fractions, one was bound to the column and the other was in the flow-through. Partial amino acid sequence determined from the lyase, retained on the anion exchange column, were identical with that of the N:-linked oligosaccharide processing enzyme glucosidase II. The lyase showed similar enzymatic properties as the microsomal glucosidase such as inhibition by 1-deoxynojirimycin and castanospermine. On the other hand, glucosidase II purified from rat liver microsomes produced not only glucose but also a small amount of 1,5-AnFru using maltose as substrate. Furthermore, CHO cells overexpressing pig liver glucosidase II showed a 1.5- to 2-fold higher lyase activity compared to the nontransfected CHO cells. Conversely, no lyase activity was detectable either in PHAR2.7, the glucosidase II-deficient mutant from a mouse lymphoma cell line, or in Saccharomyces cerevisiae strain YG427 having the glucosidase II gene disrupted. These data demonstrate that glucosidase II possesses an additional enzymatic activity of releasing 1,5-AnFru from maltose.

1,5-Anhydroglucitol promotes glycogenolysis in Escherichia coli.

Glycogen is a storage compound that provides both carbon and energy, but the mechanism for the regulation of its metabolism has not been fully clarified. Recently, we found a new glycogenolytic pathway in rat liver in which glycogen is first metabolized to 1, 5-anhydrofructose (AnFru) and then to 1,5-anhydroglucitol (AnGlc-ol). Because the amounts of glycogen and AnFru are closely related in various rat organs and the second reaction, AnFru to AnGlc-ol, is strongly inhibited in the presence of glucose, we expected that this pathway might play a regulatory role in glycogen metabolism. Here we evaluate the expected involvement of AnGlc-ol and AnFru in the regulatory mechanism in Escherichia coli C600. Having established the presence of this new glycogenolytic pathway in E. coli C600, we further show that the conversion of AnFru to AnGlc-ol is activated only after the exhaustion of glucose in the medium, and that as little as 5 microM AnGlc-ol in the medium acutely accelerates the degradation of glycogen by 40%. We consider the role of AnGlc-ol in the mechanism that controls glycogen metabolism in E. coli to be as follows. When glucose is abundant, E. coli accumulate glycogen, a fraction of which is steadily degraded so that the amount of AnFru is about 1/1,000 of glycogen on a weight basis. When glucose is depleted and the demand for glycogen utilization is elevated, AnFru, which has accumulated mostly in the medium, is promptly taken up and converted to AnGlc-ol, which accelerates glycogen degradation. We also suggest the possibility that AnGlc-ol is one of the extracellular signaling molecules for bacteria.

Purification and some properties of a hepatic NADPH-dependent reductase that specifically acts on 1,5-anhydro-D-fructose.

Glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is then reduced to 1,5-anhydro-D-glucitol (AG) in animal livers. An enzyme that catalyzes NADPH-dependent reduction of AF to AG was isolated and purified to homogeneity from porcine liver. Its apparent molecular mass was about 38 kDa on the basis of SDS-PAGE, and its monomeric dispersion in aqueous solution was indicated by gel filtration on a Superose 12 column. Amino acid sequences were determined for four peptides obtained from the purified enzyme. The resulting sequences covered about 50% of the whole sequence and indicated a remarkable similarity between the enzyme and aldose reductase. The purified enzyme showed molecular activity of 8.7 s(-1) on the basis of a molecular mass of 38 kDa, and a Km value of 0.44 mM for AF at the optimum pH of 7.0. It reduced pyridine-3-aldehyde and 2,3-butanedione effectively, acetaldehyde, glucosone, and glucuronic acid poorly and showed no detectable action on glucose, mannose and fructose. It was inactivated by p-chloromercuribenzoic acid to a considerable extent, and the inactivation was partially reversed by 2-mercaptoethanol treatment. It was also sparingly inhibited by relatively high concentrations of glucose, glucose-1(6)-phosphate and 1,5-anhydroglucitol. The reverse reaction, i.e., NADP+-dependent AG oxidation, was not observed. The observed catalytic properties and partial amino acid sequences rule out the possibility that the isolated protein is identical with any known reductase.

Hepatic production of 1,5-anhydrofructose and 1,5-anhydroglucitol in rat by the third glycogenolytic pathway.

A unique anhydrohexulose, 1,5-anhydrofructose (1,5AnFru) has been detected in rat livers. Here we describe a microanalytical method for 1,5AnFru using GC/MS and report results on the distribution and production of 1,5 AnFru in rats. The highest levels of 1,5AnFru were found in the liver (0.43 microgram/g wet tissue) and appreciable amounts were detected in adrenal gland and spleen (0.12 microgram/g and 0.09 microgram/g, respectively). Other organs contained lower amounts while plasma contained virtually no detectable 1,5AnFru. We also demonstrated that 1,5AnFru is produced in the cytosol fraction of rat liver homogenate when an alpha-1,4-glucan or glycogen was added; 1,5AnFru was readily reduced to 1,5-anhydroglucitol with NADPH or at a reduced efficiency with NADH in the presence of a Mono Q chromatographic fraction obtained from the same cytosol preparation. Based on these results, we propose the existence of a third degradation pathway, in addition to the phosphorolytic and hydrolytic reaction sequences, from glycogen to 1,5-anhydroglucitol via 1,5AnFru in mammals. However, the physiological significance of 1,5AnFru and this putative minor glycogenolytic pathway in mammals remains obscure.

Production of 1,5-anhydroglucitol from 1,5-anhydrofructose in erythroleukemia cells.

The pyranoid polyol 1,5-anhydroglucitol (1,5AnGlc-ol) occurs in a wide variety of organisms. In humans, it is present as one of the major monosaccharide components in body fluids and serves as an indicator for glycemic control in diabetic care. However, its metabolic origin and fate have been poorly understood. Here we demonstrate that 1,5AnGlc-ol is produced from glucose in erythroleukemia cells, K-562. We show the occurrence of 1,5-anhydrofructose (1,5AnFru), a derivative of 1,5AnGlc-ol oxidized at the C2 position, in K-562 cells. In addition, several pieces of evidence indicated that 1,5AnFru, rather than glucose, was the immediate precursor in 1,5AnGlc-ol production in erythroleukemia cells: exogenous 1,5AnFru was readily taken up into the cells and reduced to 1,5AnGlc-ol, but the reverse reaction, oxidation of 1,5AnGlc-ol to 1,5AnFru, was scarcely observed. The apparent K(m) of the overall cellular reduction for 1,5AnFru was estimated as 70 mg/l. This reduction was markedly inhibited by glucose in the culture medium but not by 1,5AnGlc-ol or glucitol. Since 1,5AnFru arises from alpha-1,4-glucans through lyase reactions in fungi and algae, we suggest the possibilities that glycogen in the precursor of 1,5AnFru and, therefore, 1,5AnGlc-ol originates from glycogen in mammals.

The pharmacodynamics of 6-(3-dimethylaminopropionyl)forskolin and a possible metabolite in beagles.

A specific gas chromatography/mass spectroscopy method with a detection limit of 0.1 ng/mL was developed for the measurement of 6-(3-dimethylaminopropionyl)forskolin (1) in beagle plasma. Using this method, plasma concentrations of 1 in beagles given pharmacologically effective intravenous doses of 1.HCl were determined. The observed maximal plasma concentrations rapidly decreased with time, and half-lives of the alpha-phases were < 9 min. Pharmacological effects of 1 on the cardiovascular parameters were simultaneously evaluated in one of the studies. Decreases of the pharmacological effects were slower than decreases in plasma concentration of 1. In addition, 6-(3-methylaminopropionyl)forskolin (N-monodemethyl 1), an expected initial metabolite of 1, was prepared and found to be as pharmacologically active as 1 in beagles. These results and others strongly suggest that a metabolite(s) of 1 contributes to the pharmacological effects of 1 in beagles.

Escherichia coli phosphorylates 1,5-Anhydroglucitol and releases 1,5-Anhydroglucitol 6-phosphate when glucose is absent in the medium.

The cyclic polyol 1,5-anhydro-D-glucitol (AG) is detected in most organisms, but little is known about its metabolism and physiological roles. Our previous study demonstrated that Escherichia coli C600 synthesizes AG when glucose is exhausted in the medium and that it temporarily releases AG into and then takes it back from the medium, thus forming a sharp peak in AG concentration in the medium a few hours after reaching stationary growth phase. The present study demonstrates that when glucose is absent in the culture medium, E. coli C600 takes up and phosphorylates AG and releases a large portion of it back into the medium in the form of a phosphate ester. [U-13C]AG was added to the medium after the exhaustion of glucose and the resulting [U-13C]AG phosphate was partially purified by several steps of anion exchange chromatography and identified as AG 6-phosphate by 13C-NMR. The identity of the phosphate ester was also confirmed by GC-MS analysis after further purification.

NMR of all-carbon-13 sugars: an application in development of an analytical method for a novel natural sugar, 1,5-anhydrofructose.

Of the all-carbon-13 compounds, glucose is one of the most easily accessible, and therefore we applied 13C-NMR technique to the metabolic study of glucose-related compounds, 1,5-anhydro-D-glucitol and 1,5-anhydro-D-fructose (AF). Applying an INADEQUATE method to the substitutes of these novel sugars fully labeled with carbon-13, we could trace out the entire carbon skeleton with high sensitivity and confirm the chemical structures of these sugars. The method also provided a much easier way to optimize the enzymatic oxidation for AF preparation: we selectively and continuously monitored the quantities, as well as their structures in aqueous solution, of the substrate and products in a noninvasive manner. Similarly relying upon information from the 13C-NMR, we developed a valuable derivatization method of AF for its GC-MS application, which was so sensitive that we were able to demonstrate the natural occurrence of AF in rat liver.

The ocular penetration of topical forskolin and its effects on intraocular pressure, aqueous flow rate and cyclic AMP level in the rabbit eye.

The ocular penetration of 14C-forskolin in suspension was studied using albino rabbits. The effects of topical forskolin suspension on cyclic AMP (cAMP) synthesis, aqueous flow and intraocular pressure (IOP) were also studied. It was shown that only 0.03% of the instilled forskolin penetrated the ocular tissue. The calculated kep value for forskolin was 0.2 X 10(-4) cm/hr. The peak concentrations were calculated to be 4 X 10(-7) mole/liter, 4.6 X 10(-7) and 2.7 X 10(-7) mole/1,000 g tissue in aqueous, iris and ciliary body, respectively, after instillation of 1% forskolin suspension. Topical 1% forskolin suspension caused cAMP increase in the aqueous humor 30 minutes after instillation, but cAMP returned to baseline level 60 minutes after instillation. The cAMP level in the ciliary body was not increased by forskolin. Aqueous flow did not change, and the IOP was slightly decreased 45 and 60 minutes after instillation of forskolin suspension. The in vivo least-effective concentration of forskolin in the ciliary epithelium was considered to be about 2.7 X 10(-7) mole/1,000 g tissue. The weak IOP lowering effect of topical forskolin suspension was considered to be due to its poor ocular penetration. However, slight modification of molecular structure might increase ocular penetration. Present results suggest only a slight increase in penetrative potential would be needed to make forskolin effective in antiglaucoma therapy.

Reduced renal reabsorption of 1,5-anhydro-D-glucitol in diabetic rats and mice.

A stable amount, approximately 60 micrograms, of 1,5-anhydro-D-glucitol (AG) was detected in the 24-h-urine of normal young rats fed ad libitum. Upon administration of streptozotocin (STZ), this amount was temporarily elevated to as much as 1.1 mg and AG was concomitantly removed from the circulation. The plasma AG level stayed almost null thereafter while the acutely elevated urinary AG excretion declined within 24 h to another stable excretion level that was three times as high as that of the untreated rats. In contrast, glucosuria developed much more slowly in the drug-treated rats. Normal rats and mice retained exogenous [14C]AG to a considerable extent and the radioactivity was distributed all over the body. Only a marginal fraction of the radioactivity was excreted as expired CO2. The radio-activities retained in the body and excreted into the urine were mostly attributed to unmetabolized AG. The observations of AG's metabolic stability and its relatively low level of leakage into urine suggested the concept of effective renal AG reabsorption. On the other hand, the rats with STZ-induced diabetes and NOD-mice with spontaneously developed diabetes retained little of the radioactive AG in their bodies; most of the injected radioactivity was recovered in the urine within 24 h. This observation was interpreted as due to reduced renal AG reabsorption in these animals. The concept of reduction in renal AG reabsorption in diabetes could account for the reduced plasma AG level generally observed in human diabetic cases.