Carvedilol induces endogenous hydrogen sulfide tissue concentration changes in various mouse organs

Carvedilol, a third generation non-selective adrenoreceptor blocker, is widely used in cardiology. Its action has been proven to reach beyond adrenergic antagonism and involves multiple biological mechanisms. The interaction between carvedilol and endogenous 'gasotransmitter' hydrogen sulfide (H2S) is unknown. The aim of the study is to assess the influence of carvedilol on the H2S tissue level in mouse brain, liver, heart and kidney. Twenty eight SJL strain female mice were administered intraperitoneal injections of 2.5 mg/kg b.w./d (group D1, n=7), 5 mg/kg b.w./d (group D2, n=7) or 10 mg/kg b.w./d of carvedilol (group D3, n=7). The control group (n=7) received physiological saline in portions of the same volume (0.2 ml). Measurements of the free tissue H2S concentrations were performed according to the modified method of Siegel. A progressive decline in H2S tissue concentration along with an increase in carvedilol dose was observed in the brain (12.5%, 13.7% and 19.6%, respectively). Only the highest carvedilol dose induced a change in H2S tissue level in the heart - an increase by 75.5%. In the liver medium and high doses of carvedilol increased the H2S level by 48.1% and 11.8%, respectively. In the kidney, group D2 showed a significant decrease of H2S tissue level (22.5%), while in the D3 group the H2S concentration increased by 12.9%. Our study has proven that carvedilol affects H2S tissue concentration in different mouse organs.     

delta13C-values of endogenous urinary steroids

By means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) urinary steroids obtained from a reference population of 56 subjects were analyzed for their (13)C/(12)C-ratios. The analytes encompassed androsterone (A), etiocholanolone (E), 11beta-hydroxyetiocholanolone (OHE), 11beta-hydroxyandrosterone (OHA), and 5beta-pregnane- 3alpha,20alpha-diol (PD). A and E represent androgen metabolites (AM). PD, OHE, and OHA have sources independent from androgen metabolism. The delta(13)C-values of the latter compounds may be compared to those of AM in order to detect doping with synthetic androgens and thus may serve as endogenous reference compounds (ERC). In order to allow for classification of conspicuous samples, reference ranges and limits were calculated for delta(13)C-values of selected steroids and differences hereof (Delta(13)C-values). When A is compared to ERCs, Delta(13)C-values larger than 3 per thousand are very unlikely. A set of additional parameters was surveyed by a questionnaire. Several factors turned out to exert significant influence on the delta(13)C-values of urinary steroids. These encompass the identity of the steroid itself, sex, oral contraception, travels, and physical activity.     

Measurement of excreted steroids in Macaca nemestrina

A practical method for the quantitative measurement of the estrogenic steroid estradiol-17β in the feces of pigtailed macaques (Macaca nemestrina) is described. The method, which includes homogenization, filtration, ether extraction, and sephadex purification, produces an 85.3% recovery of 3H-estradiol. Comparable recovery of 3H-progesterone (64.1%) and 3H-testoster-one (67.9%) is also obtained. Estradiol, measured by radioimmunoassay methodology, has inter- and intra-assay variations of 7.1% and 4.3%, respectively. Linearity of estradiol was checked by varying the amount of feces analyzed. Results show a correlation of 0.9945, Y = 32.526x + 0.205. Accuracy of estradiol, determined by adding radioinert estradiol to the feces before homogenization, was 98.7% with a correlation of 0.9950, Y = 1.027x ? 0.543. The menstrual cycles of several animals were analyzed and corresponded well with serum levels of estradiol-17β. Similar correspondence was obtained in matched fecal and serum samples from pregnant animals. These methods provide a practical and accurate solution to the problem of collecting hormone data in field studies without the potential complications of capture in laboratory research.     

Quantitative assessment of endogenous testicular and adrenal sex steroids and of steroid metabolizing enzymes in untreated human prostatic cancerous tissue

Total tissue content and subcellular distribution of DHEA sulfate, DHEA, androst-5-ene-3 beta,17 beta-diol, androst-4-ene-3,17-dione, testosterone, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol as well as the activities of steroid sulfate-sulfatase, 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenase, and creatine kinase were quantified in 12 untreated primary tumors of prostatic cancer. Samples were obtained by radical prostatectomy and serial sections, and were alternately used for either biochemical or morphological evaluation. The results were compared with values determined in benign parts of the same prostates. Qualitatively, all enzymes and steroids found in the benign tissues could also be demonstrated in the cancers. Steroid patterns showed individual quantitative variation but no general differences between the carcinomas and the benign tissues. Enzymes showed a tendency to lower activities in the cancers, particularly when expressed per DNA. Substantial diminutions of creatine kinase and 5 alpha-reductase activity, the latter being often accompanied by an increased testosterone/DHT ratio, were the most striking differences seen in most of the cases between malignant and nonmalignant tissues. Some interesting individual parallels of morphological and biochemical aspects were seen, but there was no obvious general parallelism between the histological picture and endocrinological characteristics.     

Evidence for role of endogenous sex steroids in morphine antinociception.

The effect of morphine on responses to sustained mild pain was tested in male white rats with gonads and (or) adrenals removed. Morphine was ineffective in gonadectomized rats; adrenalectomy alone increased the effectiveness of morphine over that in sham-operated controls. Morphine antinociception may involve some actions of endogenous steroids.     

Measurement of endogenous alloxan in human blood

For determination of endogenous alloxan content in blood of healthy donors it was stabilized by rapid lowering pH in a sample to 2.0. Alloxan was then allowed to form a colored product in a reaction with [ital]o-phenylenediamine and its content was measured by spectrophotometry using an internal calibration curve. Analysis of 75 blood samples of healthy volunteers has shown that in most donors alloxan concentrations vary from 41 to 265 μmol/l. However, in a small group of healthy people alloxan levels greatly exceeded the above-mentioned limit.     

Levels and subcellular distribution of endogenous corticosterone in rat liver during development

The concentration of corticosterone in liver homogenates, liver cytosol and purified nuclear fractions, and in plasma of fetal, newborn, immature and adult rats has been measured by radioimmunoassay.Highest plasma corticosterone levels were found in fetal rats, decreasing close to the levels observed in the adrenalectomized rat by the 6th day of postnatal life followed by a rise in the adult rat. The concentration of corticosterone in liver during development paralleled the plasma levels, the liver to plasma corticosterone ratio ranging between 0.09 and 0.17 suggesting that the corticosterone retained by the tissue is related to the unbound fraction of the hormone in plasma.Both plasma and tissue corticosterone levels declined after adrenalectomy and they were elevated after ether stress.Fractionation of liver homogenates showed that the major fraction of liver corticosterone is localized in the cytosol. Purified liver nuclei contained between 9 and 16% of the total liver corticosterone. The amount of corticosterone in the nuclei seems to be related to the plasma and tissue hormone levels rather than the concentration of cytoplasmic glucocorticoid receptors. Since most of the nuclear corticosterone appears to be bound to receptors, it has been calculated that close to 60% of the cellular receptors in fetal liver are localized in the nucleus. In adult rat liver, only about 10% of the cellular receptors appear to be associated with nuclei. Changes in the concentration of glucocorticoid receptors in liver during development and after adrenalectomy are inversely related to changes in plasma corticosterone levels. It is suggested that corticosterone may regulate the levels of its own receptors in liver.     

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.     

Feasibility of the immunohistochemical detection of endogenous steroids in paraffin-embedded ovarian tumours

Summary Formalin-fixed, paraffin-embedded samples from 25 ovarian granulosa cell tumours and six poorly differentiated ovarian carcinomas examined immunohistochemically for the presence of estradiol, testosterone and progesterone. Twenty-four of the 25 granulosa cell tumours stained for estradiol predominantly in granulosa cells and also in the theca cells, but none of the carcinomas were positive. All the granulosa cell tumours and five out of six carcinomas were positive for progesterone, while 13 granulosa cell tumours and three carcinomas stained moderately positive for testosterone.No clear-cut relationship was observed between positivity for estradiol in granulosa cell tumours and occurrence of endometrial hyperplasia. In two cases of granulosa cell tumours, 1–15% cells showed ultrastructural features of steroid synthesis: i.e. moderate to abundant smooth endoplasmic reticulum and mitochondria with tubular cristae. This is in contrast with the diffuse immunohistochemical staining reaction of granulosa cell tumours with antisteroid antibodies.To evalutate the significance of immunohistochemical for estradiol, four estradiol-containing tumours were tested by radioimmunoassay for the presence of estradiol before and after dehydration. As expected, treatment with organic solvent leads to a drastic reduction in estradiol content below the detection level of the assay. The result indicate that immunohistochemical staining for steroids of paraffin-embedded granulosa cell tumours if of limited value.     

The subcellular distribution of endogenous and exogenous serotonin in brain tissue: comparison of synaptosomes storing serotonin, norepinephrine, and gamma-aminobutyric acid

Abstract— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [³H]5-HT (5 × 10^-8 M), for 45 min, when there was apparent equilibrium between [³H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [³H]5-HT in sucrose gradients was separable from the peak for [¹⁴C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [³H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

Changed levels of endogenous sex steroids in women on oral contraceptives.

Serum and urinary levels of unconjugated testosterone, dihydrotestosterone, and oestradiol were measured by specific radioimmunoassays in 10 healthy women in the early follicular phase of their menstrual cycle and in nine healthy women taking oral contraceptives. The contraceptive group had testosterone levels 1-3 times higher and dihydrotestosterone levels two times higher than those in the controls. Serum oestradiol levels in the contraceptive group were much lower than those in the controls and similar to levels in postmenopausal women. The contraceptive group had about twice the urinary excretion of unconjugated (free) testosterone and dihydrotestosterone of the controls, but their excretion of unconjugated oestradiol was 2-7 times lower. The great increase in serum and urinary androgen concentrations, as well as the suppression of oestradiol, may be related to the antiovulatory effect of oral contraceptives.     

Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants

The cellular and subcellular localization of endogenous nitric oxide (NO.) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO. generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)(2) and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO. was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO., with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO. was also found in these organelles, and the level measured of NO. was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO. from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO. mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH(4); and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO. could be involved in the process of senescence of pea leaves.