Bioconversion of isosorbide dinitrate by various microorganisms

Summary A total of 19 microorganisms, selected from genera of bacteria, fungi and yeasts, were screened for their ability to hydrolyse isosorbide dinitrate (ISDN) to mononitrates. Cunninghamella echinulata and Cunninghamella elegans showed rates of bioconversion of ISDN of 74% and 88% respectively, measured after 73 h. However, the two strains exhibited opposite stereoselectivity, as reflected in the ratios of isosorbide 5-mononitrate (5-ISMN) to isosorbide 2-mononitrate (2-ISMN). These were 2.57 and 0.75 for C. echinulata and C. elegans, respectively.     

Patterns of isosorbide dinitrate and glyceryl trinitrate metabolites formed by selected segments of the rabbit gastrointestinal tract

Homogenates of selected segments of the rabbit gastrointestinal tract (GIT) were studied for their ability to biotransform isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) to their mono- and di-nitrate metabolites, respectively. In addition, preferential formation of certain metabolites was investigated by examination of the patterns of metabolites formed by the various homogenates. After a 30-min incubation of ISDN with GIT homogenates (pH 7.4, 37 degrees C), the percent disappearance of ISDN and the ratio of isosorbide-2-mononitrate (2-ISMN) to isosorbide-5-mononitrate (5-ISMN) were as follows: stomach, 32%, 0.8; duodenum, 65%, 0.1; jejunum, 59%, 0.2; ileum, 38% , 1.2; cecum, 33%, 2.7; and colon, 32%, 3.4. After a 5-min incubation of GTN with GIT homogenates, the percent disappearance of GTN and the ratio of glyceryl-1,3-dinitrate (1,3-GDN) to glyceryl-1,2-dinitrate (1,2-GDN) were as follows: duodenum, 54%, 0.65; ileum, 73%, 0.68; and colon, 61%, 0.17. Incubation of 2 x 10(-7) M ISDN with mucosal and muscularis homogenates of duodenum, jejunum, and ileum resulted in significant losses of ISDN with an equimolar formation of the mononitrate metabolites. Most of the metabolic activity for ISDN resided in the mucosal layer of each section. The ratio of 2-ISMN to 5-ISMN varied in each section (stomach to colon) and cross section (mucosal versus muscularis) of the GIT. We conclude that the metabolism of ISDN and GTN by the GIT may contribute to the high clearance of these organic nitrates, and the low oral bioavailability of ISDN. Also, multiple mechanisms appear to be involved in the biotransformation of ISDN and GTN in the rabbit GIT.     

Differential biotransformation of the enantiomers of isoidide dinitrate in isolated rat aorta

Previous studies have demonstrated that the D-enantiomer of isoidide dinitrate (IIDN) is 10-fold more potent than the L-enantiomer for relaxation and cyclic GMP accumulation in isolated rat aorta. To test whether preferential biotransformation of D-IIDN to a species that activates guanylate cyclase is the basis for this observed enantioselectivity, paired segments of rat aorta were exposed to D- and L-IIDN and the tissue accumulation of the parent compound and the formation of their respective metabolites (D- and L-isoidide mononitrate, IIMN) were determined. The extent of relaxation of rat aorta following exposure to 2 microM D-IIDN was greater than that by L-IIDN over a 5-minute time course, and this was associated with a higher rate of D-IIDN biotransformation to D-IIMN at all time points. In addition, the rate of D-IIDN biotransformation was greater than that of L-IIDN at most IIDN concentrations tested. By contrast, the amount of D- and L-IIDN in the tissue was the same at all time points and concentrations tested, indicating that selective uptake of D-IIDN into blood vessels did not occur. When tissues were made tolerant to organic nitrate-induced relaxation by treatment with a high concentration of glyceryl trinitrate, the biotransformation of both D- and L-IIDN was attenuated. This suggests that mechanism-based biotransformation may be affected during tolerance development. Furthermore, the association of preferential D-IIDN biotransformation with its greater potency for vasodilation and cyclic GMP accumulation suggests than an enantioselective site for biotransformation is an important component of organic nitrate-induced vasodilation.     

Pulmonary biotransformation of insulin in rat and rabbit.

In vitro biodegradation of insulin in rabbit and rat lung homogenates was investigated. Insulin can be sequentially metabolized into two primary fragments in rabbit lung homogenate by an aminopeptidase. The amino acid sequences of the fragments were found to be the des-Phe-InsulinB1 (Metabolite I) and des-Phe-Val-InsulinB1-2 (Metabolite II). However, only the former metabolite (Metabolite I) was identified in the rat lung homogenate. The km and Vm values associated with rabbit lung homogenate were 0.29 +/- 0.14 mM and 16.4 +/- 6.9 microM/hr/mg protein, respectively, whereas those for a rabbit lung preparation containing both microsomes and cytosol were 0.22 +/- 0.07 mM and 17.9 +/- 5.4 microM/hr/mg protein, respectively. The km and Vm associated with the cytosolic fraction of rabbit lung were 0.32 +/- 0.16 and 20.6 +/- 6.1 microM/hr/mg protein, respectively. The results indicate that the lung aminopeptidase may be a cytosolic enzyme. The degradation of dimeric insulin in the lung homogenate was faster than that of hexameric insulin due to the difference in collision frequency between the enzyme and insulin aggregates. The major metabolites in the lungs reportedly retain almost the same bioactivity of insulin, suggesting that the pulmonary route of insulin delivery will not adversely affect its hypoglycemic activity.     

Bioconversion of isosorbide dinitrate into isosorbide mononitrate by the protozoan Tetrahymena thermophila: relationship to glutathione transferase levels

Summary Growing cells of Tetrahymena thermophila (T. t.) metabolized, after 72 h, 80% of isosorbide dinitrate (ISDN) to isosorbide 5-mononitrate (5-ISMN) and isosorbide 2-mononitrate (2-ISMN) in a ratio 5-ISMN/2-ISMN=2.6 as evaluated by HPLC. The level of glutathione S-transferases (GSH-ST) determined by following the reactions with of 1-chloro-2,4-dinitrobenzene (CDNB), o-dinitrobenzene (o-DNB) or ISDN, showed the inductive effect of ISDN (0.5 mg/ml) on the level of this enzyme. The enzymatic activity, evaluated at 72 h, showed a twofold increase compared with the control. The GSH-ST activity correlated well with the rate of ISDN bioconversion.     

Gas chromatographic determination of isosorbide dinitrate in human plasma and urine

A rapid, simple and sensitive method for the specific determination of isosorbide dinitrate concentrations down to 0.5 ng/ml in human plasma and urine is described. Following traction (with or without internal standard) of isosorbide dinitrate into toluene, the compound is determined by gas chromatography using a ⁶³Ni electron-capture detector.     

Structural and functional differences in various divisions of the rabbit colon

Summary The rabbit colon displays a diversity of form and function along its proximo-distal axis. Morphologically, four regions can be discerned based on macroscopic and microscopic criteria: 1) the initial portion of the colon immediately distal to the cecum (P1), in which wart-like protrusions characterize the surface topography, is 10cm in length and endowed with three teniae. 2) The adjoining portion of the colon (P2) possesses one tenia, is about 20cm in length and also displays the wart-like protrusions in slightly less prominent form. 3) Fusus coli, a short segment approximately 4 cm in length, is free of teniae, but exhibits longitudinal folds on its inner aspect. Electron microscopically, it shows a paucity of microvilli in direct contrast to the two afore-mentioned regions. These three portions together constitute the proximal colon. 4) The fourth region of the colon, the distal colon, reaches a length of 80–100cm and shows no obvious second-order enlargements of its surface, displaying scanning electron microscopically ridges in looped configurations. Physiological parameters also showed differences depending on the region of the colon observed. Water content of the ingesta increases slightly during passage of the proximal colon, decreasing in the fusus coli and distal colon. Na concentration was highest in the area P1–P2, decreasing distally. K was low in area P2 and then rose toward the fusus, only to fall again distally. Nitrogen values decrease considerably during passage of P2 but only slightly distally. Transmural electrical potential differences also exhibit a characteristic, discontinuous gradient.     

Sex differences in the biotransformation of 2-acetylaminofluorene in cultured rat hepatocytes

Sex-related differences in susceptability to 2-acetylaminofluorene (2-AAF) hepatocarcinogenicity and in vivo biotransformation of 2-AAF have been observed. In order to determine the contribution of hepatocytes to these differences, 2-AAF biotransformation was investigated in monolayer cultures of hepatocytes freshly isolated from male and female F-344 rats.In cultured hepatocytes from both sexes, ring and N-hydroxylated, deacetylated and conjugated metabolites were formed. The half-life of 2-AAF was similar at concentrations of 5×10^–6 and 10^–5 M; however, at 10^–4 M a slower rate was observed in cultures from males. Although the total formation of aqueous metabolites was similar, the ratio of sulfate to glucuronide conjugates of 2-AAF formed by hepatocytes from male and female rats differed. Sulfate conjugates predominated in hepatocytes from male rats, whereas in females, glucuronides predominated. The demonstration of sex-dependent variations in the rate of metabolism at a high concentration of 2-AAF and in conjugation provides evidence that in vivo differences are a function, at least in part, of the biotransformation characteristics of hepatocytes.Abbreviations 2-AAF 2-acetylaminofluorene - 2AF 2-aminofluorene - WME Williams Medium E.     

Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes

Since nitric oxide (NO) in many cells is involved in energy metabolism, the aim of this study was to evaluate the role of isosorbide dinitrate (ISDN), a NO donor, in energy metabolism of rat reticulocytes, particularly due to their high content of hemoglobin--an effective scavenger of NO. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated in the absence (control) or in the presence of different concentrations of ISDN. ISDN decreased total and coupled oxygen consumption (p<0.05) while increased uncoupled oxygen consumption (p<0.05) in a dose- and time-dependent manner. This was followed by enhancement of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.05). Levels of all glycolytic intermediates in the presence of ISDN indicate only stimulation of pyruvate kinase activity. ISDN did not alter the concentration of ATP, while increased ADP and AMP levels (p>0.05). In rat reticulocytes under steady-state conditions, 95.4% of overall energy was produced by oxidative phosphorylation but only 4.6% by glycolysis. Due to a reduced coupled oxygen consumption in the presence of ISDN, ATP production via oxidative phosphorylation was significantly diminished. A simultaneous increase of glycolytic ATP production is not enough to ensure constant ATP production. The calculated mean ATP turnover time was prolonged by 199% in the presence of 1.5 mmol/l ISDN. In conclusion, ISDN a) inhibited total and coupled respiration but enhanced uncoupled respiration, b) stimulated glycolysis, c) decreased ATP production and d) prolonged ATP turnover time in rat reticulocytes. These effects were mediated by NO as the effector molecule.     

Immunohistochemical demonstration of prostaglandins in various tissues of the rat

To demonstrate the tissue localization of prostaglandin (PG) E₂, PGF₂ and 6-keto-PGF₁ (a stable metabolite of PGI₂) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE₂-, PGF₂-, and 6-keto-PGF₁-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF₁ was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE₂ or PGF₂ was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.     

Sex- and lineage-specific inheritance of depression-like behavior in the rat

The Wistar–Kyoto (WKY) rat exhibits physiological and behavioral similarities to endophenotypes of human depression. In the forced swim test (FST), a well-characterized antidepressant-reversible test for behavioral despair in rodents, WKYs express characteristics of behavioral despair; increased immobility, and decreased climbing. To map genetic loci linked to behavior in the FST, we conducted a quantitative trait loci (QTL) analysis of the segregating F2 generation of a WKY × Fisher 344 (F344) reciprocal intercross. Using linear-model-based genome scans to include covariate (sex or lineage)-by-QTL interaction effects, four significant QTL influencing climbing behavior were identified. In addition, we identified three, seven, and two suggestive QTL for climbing, immobility, and swimming, respectively. One of these loci was pleiotropic, affecting both immobility and climbing. As found in human linkage studies, several of these QTL showed sex- and/or lineage-dependent effects. A simultaneous search strategy identified three epistatic locus pairs for climbing. Multiple regression analysis was employed to characterize the joint contributions of these QTL and to clarify the sex- and lineage-dependent effects. As expected for complex traits, FST behavior is influenced by multiple QTL of small effect, each contributing 5%–10%, accounting for a total 10%–30% of the phenotypic variance. A number of loci mapped in this study share overlapping candidate regions with previously identified emotionality QTL in mice as well as with susceptibility loci recognized by linkage or genome scan analyses for major depression or bipolar disorder in humans. The presence of these loci across species suggests that these QTL may represent universal genetic factors contributing to mood disorders.     

Comparison of the rat microsomal Mg-ATPase of various tissues

The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.     

Sex- and age-related differences in arterial pressure and albuminuria in mice

Background

Animal models have become valuable experimental tools for understanding the pathophysiology and therapeutic interventions in cardiovascular disease. Yet to date, few studies document the age- and sex-related differences in arterial pressure, circadian rhythm, and renal function in normotensive mice under basal conditions, across the life span. We hypothesized that mice display similar sex- and age-related differences in arterial pressure and renal function to humans.

Methods

Mean arterial pressure (MAP) and circadian rhythm of arterial pressure were measured over 3 days via radiotelemetry, in 3- and 5-month-old (adult) and 14- and 18-month-old (aged) FVB/N and in 5-month-old (adult) C57BL/6 male and female normotensive mice. In FVB/N mice, albuminuria from 24-h urine samples as well as body, heart, and kidney weights were measured at each age.

Results

Twenty-four-hour MAP was greater in males than females at 3, 5, and 14 months of age. A similar sex difference in arterial pressure was observed in C57BL/6 mice at 5 months of age. In FVB/N mice, 24-h MAP increased with age, with females displaying a greater increase between 3 and 18 months of age than males, such that MAP was no longer different between the sexes at 18 months of age. A circadian pattern was observed in arterial pressure, heart rate, and locomotor activity, with values for each greater during the active (night/dark) than the inactive (day/light) period. The night-day dip in MAP was greater in males and increased with age in both sexes. Albuminuria was greater in males than females, increased with age in both sexes, and rose to a greater level in males than females at 18 months of age.

Conclusions

Arterial pressure and albuminuria increase in an age- and sex-specific manner in mice, similar to patterns observed in humans. Thus, mice represent a useful model for studying age and sex differences in the regulation of arterial pressure and renal disease. Understanding the mechanisms that underlie the pathophysiology of cardiovascular disease may lead to new and better-tailored therapies for men and women.

     

Properties and concentration of somatomedin A in various rat tissues

The concentration of somatomedin A (SMA) in various rat tissues was determined directly by the radioreceptor assay in supernates of tissue homogenates at 100,000 x g for 60 min. The SMA concentrations in pancreas, kidney, lung, liver, spleen, testis, brain and muscle were 2.56, 2.40, 2.28, 2.16, 1.56, 1.44, 0.60 and 0.30 U/g of wet tissue, respectively. The SMA content in rat serum was 6.66 U/ml. The SMA concentration in tissues never exceeded that in serum. When the 100,000 x g pellet of liver was treated with a hypotonic solution, the SMA content in the hypotonic solution was about 35% of that in the initial supernate. This result indicates that about 30% of the SMA in liver was stored in organellae. SMA in tissues decreased with incubation at 37 degrees C, though SMA in serum did not. This decrease could be precluded by adding 0.75 mM HgCl2 to the liver homogenate. The SMA content of the liver homogenate was dependent on growth hormone in experiments both in vivo and in vitro.     

The effects of isosorbide dinitrate on methemoglobin reductase enzyme activity and antioxidant states

Isosorbide dinitrate (ISDN) has been used in the treatment of ischaemic cardiovascular diseases for many years. ISDN is the most popular nitric oxide donor and causes methemoglobinemia as an important side-effect. The purpose of this study was to examine antioxidant states and methemoglobin reductase activity after giving ISDN and ISDN plus vitamin E. Rats were divided into three groups according to the treatment: control group, ISDN group and ISDN plus vit. E group. We measured reduced glutathione in blood (GSH), plasma malondialdehyde (MDA), erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and NADH-dependent methemoglobin reductase activities. In the ISDN group, plasma MDA levels were significantly high compared to the control and ISDN + vit. E groups (p < 0.001). In the ISDN and ISDN + vit. E groups, blood GSH levels were higher than those of the control group (p < 0.05). Changes of SOD and GPx activities were not significant. In the ISDN and ISDN + vit. E groups the erythrocyte catalase and NADH-dependent methemoglobin reductase activities were significantly higher than that in the control group (p < 0.001). We conclude that oxidant drugs such as ISDN need to be carefully used because of lipid peroxidation and methemoglobinemia. These findings support the notion that vitamine E protects tissues against oxidative stress.