Biomedical subjects

R Gossrau

Publications and source records attributed to R Gossrau.

At least 37 records · Page 2Linked to original sources

PubMed1996

Nitric oxide synthase I immunoreactivity and NOS-associated NADPHd histochemistry in the visceral epithelial cells of the intraplacental mouse yolk sac.

In the course of our studies on the local blood flow modulation in the NMRI-mouse placenta we have focussed on regulatory pathways involving recently appreciated gaseous messenger molecules nitric oxide (NO) and carbon monoxide (CO), which are generated by NO synthase (NOS) and heme oxygenase (HO)-2, respectively. The distribution of NOS was investigated by immunohistochemistry using an antiserum to the neuronal isoform (NOS-I) and by NADPH diaphorase (NADPHd) histochemistry, supplemented with procedures (permanganate and formaldehyde method) serving to enhance the specificity of the enzyme histochemical method for NOS visualization. HO-2 was demonstrated immunohistochemically. In addition, cyclic guanosine monophosphate (cGMP)-forming soluble guanylate cyclase (sGC) and dehydrogenases generating the NOS co-substrate NADPH were analysed either by immunohistochemistry or enzyme histochemistry. NOS-I immunostaining was observed in the intraplacental visceral yolk sac epithelial cells but not in the placenta and extraplacental visceral epithelial yolk sac cells. Co-localization of NOS-I immunolabeling and NOS-associated NADPHd was exclusively found in the intraplacental visceral epithelial cells, while NADPHd activity not associated to NOS was present in other placental and extraplacental cells additionally analysed for control reasons. HO-2 and sGC immunoreactivity could not be detected in the placenta including the intraplacental visceral epithelial cells but were expressed in several extraplacental cells. Dehydrogenases producing the NOS co-substrate NADPH were present in the intraplacental visceral epithelium as well as in other placental and extraplacental cells. Since the intraplacental visceral epithelial yolk sac layer closely accompanies large fetal blood vessels entering the placental labyrinth from the chorionic plate it may be assumed that NO, generated by the NADPH-consuming NOS-I in the intraplacental yolk sac epithelium, acts to regulate the blood flow by relaxing smooth muscle cells in the wall of these fetal vessels. The lack of immunoreactivity to the NO-effector molecule sGC may be due to methodological reasons. The absence of the HO-2/CO system suggests its insignificant role as a potential gas signaling pathway in the vascular smooth muscle system of the intraplacental visceral yolk sac of mice.

Animals↗

PubMed1996

Expression of heme oxygenase-2 (HO-2)-like immunoreactivity in rat tissues.

Microsomal heme oxygenase (HO) is a cytochrome P-450-assisted oxidoreductase, which catalyzes the NADPH-dependent decomposition of heme to carbon monoxide (CO), biliverdin, and iron. Recent evidence suggests that CO, similar to nitric oxide (NO), may serve as gaseous biological signalling molecule, which acts by stimulating soluble guanylate cyclase in target cells. In the present investigation, we report the HO-like immunoreactivity (LIR) pattern of the constitutive HO isozyme, HO-2, and compare the results with recently published data on constitutive NO-producing nitric oxide synthase (NOS) in rat tissues. HO-2-LIR was most consistently observed in connective tissue elements (fibrocytes/-blasts and fibroblast-like cells, such as interstitial cells in the bowel), blood vessel wall constituents (arterial and venous endothelial cells, vascular smooth muscle cells), visceral smooth muscle cells (airway musculature, myometrium, muscularis mucosae of the small intestine), mesothelial cells of serous membranes and in select epithelial cell populations. HO-2-LIR was absent from the striated (skeletal and cardiac) musculature. HO-2 had a more widespread distribution and its expression largely differs from that of NOS. HO-2-LIR and NOS appear to be co-expressed in vascular endothelial cells and in selected nerve cell populations of certain parasympathetic and probably sensory ganglia. Our data suggest potential CO and NO systems as interrelated regulatory pathways in the local paracrine and autocrine control of diverse functional systems.

Animals↗

PubMed1996

Nitric oxide synthase I (NOS-I) is deficient in the sarcolemma of striated muscle fibers in patients with Duchenne muscular dystrophy, suggesting an association with dystrophin.

Previously, we have demonstrated the expression of the brain-type nitric oxide synthase (NOS-I) in the sarcolemmal region of somatic and visceral striated muscle fibers in a variety of mammalian species through the use of enzyme histochemical and immunochemical techniques. Here we report that NOS-I protein and its NADPH diaphorase (NADPHd) activity are co-localized in the sarcolemma of human skeletal muscles. NOS-I immunolabeling and NADPHd activity showed no significant variation between type I and II fibers. In muscle biopsy specimens from patients with Duchenne muscular dystrophy (DMD), both NOS-I protein and activity were absent or markedly reduced. We, therefore, propose that NOS-I is complexed with dystrophin and/or dystrophin-associated proteins, adding a novel member to the sarcolemmal dystrophin-glycoprotein complex (DGC). The nature of the NOS-I-DGC link, and its role in skeletal muscle physiology and pathophysiology remain to be elucidated.

Child↗

PubMed1995

Distribution and quantification of alpha 1-integrin subunit in rat organs.

The alpha 1 beta 1-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the alpha 1-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the alpha 1-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the alpha 1-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the alpha 1-integrin subunit, respectively, enabled the quantitative expression pattern of the alpha 1-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the alpha 1-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of alpha 1-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of alpha 1-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the alpha 1-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell-collagen binding.

Animals↗

PubMed1995

Species-independent expression of nitric oxide synthase in the sarcolemma region of visceral and somatic striated muscle fibers.

The expression and distribution of nitric oxide synthase (NOS) was studied by use of the newly designed specific histochemical NADPH diaphorase staining method and the indirect immunofluorescence technique employing an antiserum to brain NOS in visceral and somatic striated muscles of several mammalian species. Histochemical activity and immunoreactivity were located in the sarcolemma region of type I and II fibers of all muscles investigated. Visceral muscles were more strongly stained than somatic muscles. Furthermore, type II fibers, identified by staining of myosin adenosine triphosphatase activity after pre-incubation at alkaline pH, were more intensely labeled than type I fibers. In addition, NOS activity was detected in the area of the sarcolemma of intrafusal fibers. No obvious differences between species were observed. It was concluded that NOS of striated muscles probably makes up the richest and most important nitric oxide source in mammals.

Animals↗

PubMed1995

Alpha-NADPH appears to be primarily oxidized by the NADPH-diaphorase activity of nitric oxide synthase (NOS).

Biochemical studies have shown that the NADPH-diaphorase (NADPH-d) activity of nitric oxide synthase (NOS) represents only a part of the total cellular diaphorase pool. Histochemically, NADPH-d activity can be demonstrated in cells expressing no constitutive NOS. Therefore, attempts aimed to improve the specificity of the NADPH-d reaction are currently being undertaken. In this study, the effect of replacing the natural and common diaphorase substrate beta-NADPH with the artificial stereoisomer alpha-NADPH on the extent of NADPH-d staining was examined. When beta-NADPH served as the substrate, discrete populations of central and peripheral neurons as well as numerous non-neural cells in many organs of common laboratory rodents (mouse, rat, gerbil, hamster, guinea pig) and marmosets were found to generate formazan. Substitution of alpha-NADPH for beta-NADPH resulted in reduced staining intensity of nerve cells and muscle fibers. Furthermore, alpha-NADPH-d staining of macula densa cells, enterocytes and granulocytes varied according to the species examined. No reaction was observed in most other cells which stained positively for beta-NADPH-d activity. Examination of adjacent sections, incubated for the demonstration of NOS-immunoreactivity, revealed that alpha-NADPH-d activity and NOS immunostaining are strictly colocalized in neurons, striated muscle fibers and, species-dependently, in macula densa cells. It can thus be concluded that, with the exception of gut granulocytes, alpha-NADPH is primarily metabolized by the reductase activity of NOS.

Animals↗

PubMed1995

Demonstration of nitric oxide synthase (NOS) in marmosets by NADPH diaphorase (NADPH-d) histochemistry and NOS immunoreactivity.

Since species interdiversity often prevents the extrapolation of laboratory rodent data to man and similar problems may exist for nitric oxide synthase (NOS), NADPH-d activity and immunohistochemistry of NOS were investigated in the New World monkey Callithrix jacchus (marmoset), which has been shown to be close to the human situation in many respects. Using the NADPHd reaction with beta-NADPH and nitroblue tetrazolium (NBT) on acetone-chloroform pretreated cryosections, NBT formazan was found in many neural and non-neural (e.g. diverse epithelia, striated muscle fibers, vascular endothelium) cells in numerous tissues and organs. Prefixation with formaldehyde lowered the number of NADPH-d active sites and the amount of formazan with the exception of neuronal NADPH-d as did incubation of fresh or acetone-chloroform-pretreated sections for NADPH-d in the presence of 0.5% formaldehyde. When 1% formaldehyde or 0.5 mM permanganate were used significant amounts of formazan appeared only in central and peripheral neurons, vasal endothelial cells, small intestinal enterocytes, plasma membrane region of striated muscle fibers as well as arteriolar cells in the kidney; except for enterocytes, these observations were confirmed by NOS-immunohistochemistry which revealed in addition reactive cells in the thymus and intestinal lamina propria.

Acetone↗

PubMed1995

Hydrogen peroxide (H2O2) production by monoamine oxidase in rat tissues using endogenous catecholamines as substrates. A comparison of catalytic monoamine oxidase histochemistry and recently published catechol-O-methyltransferase immunohistochemistry.

Histochemical studies on hydrogen peroxide (H2O2) production by monoamine oxidase (MAOX) using xenobiotic (foreign) catecholamines such as tryptamine or tyramine as substrates may not reveal the true H2O2-production capacity of this enzyme and the potential co-localization and cooperation of MAOX with catechol-O-methyltransferase (COMT), the other catecholamine-degrading enzyme. Therefore, in the present study the catecholamine hormones adrenaline (epinephrine) and noradrenaline (norepinephrine) and the catecholamine neurotransmitter noradrenaline as well as the COMT metabolites metanephrine and normetanephrine, which are likely to be the more important MAOX substrates, were used for MAOX visualization in many rat tissues with a cerium-diaminobenzidine-H2O2-Co method. Adrenaline and noradrenaline were autooxidized by Ce3+ and could not be employed; with metanephrine or normetanephrine as substrates MAOX produced considerable amounts of H2O2 in many cells and tissues. Comparisons with immunohistochemical COMT-data for rats from the literature show that MAOX and COMT are co-localized or not. Therefore, different from our current knowledge in rats COMT and MAOX either co-operate in catecholamine degradation or they degrade the respective catecholamines alone.

Animals↗

PubMed1995

Visualization of hydrogen peroxide (H2O2)-production from histamine.

The cellular and intracellular metabolization sites of the tissue hormone and paracrine compound histamine as a source for the indirect and potentially toxic or physiological mediator molecule H2O2 are not yet known. Therefore, in the present study, histamine was used as the substrate in a cerium-diaminobenzidine-H2O2-Co procedure to visualize for the first time the oxidative deamination and H2O2-production sites of this amine in various laboratory animals. Diamine oxidase (DAOX) was shown to be the responsible enzyme. With the exception of marmosets, all species could deaminate histamine oxidatively and form H2O2. In most species, H2O2 was produced by DAOX from histamine in small intestinal enterocytes; in rats H2O2 was generated in all vascular and non-vascular smooth muscle cells; in guinea-pigs only smooth muscle cells in the digestive tract and uterus and in addition the cardiac and gastric capillary endothelium and hepatic sinusoidal endothelium produced H2O2. Furthermore, in some species H2O2 was generated by DAOX with histamine as the substrate in certain renal, adrenal and splenic cell types. While H2O2-production in enterocytes may derive from luminal-borne histamine, i.e., from histamine of foreign origin, the formation of H2O2 in the other cells suggests endogenous (mast cell, basophilborne) histamine as the substrate and H2O2 source.

Amine Oxidase (Copper-Containing)↗

PubMed1995

A modified method allows for correlation between NADPH-diaphorase histochemistry and immunohistochemistry for the demonstration of neuronal nitric oxide synthase (nNOS).

Results obtained with the conventional nitro blue tetrazolium salt method for the visualization of the NADPH-diaphorase (NADPH-d) activity of nitric oxide synthase (NOS) are not specific for this particular enzyme, since this activity represents only a fraction of the total cellular NADPH-d pool. Therefore, the standard NADPH-d procedure was modified by performing the incubation in the presence of formaldehyde. Parallel application of the modified NADPH-d staining technique and the indirect immunofluorescence using an antibody against the neuronal isoenzyme (nNOS) on rat, mouse and guinea-pig tissues showed a correlation between histochemical and immunocytochemical staining. It can thus be concluded that the modified NADPH-d procedure allows for a more specific detection of the histochemical nNOS activity than the conventional method.

Animals↗

PubMed1995

Nonspecific alkaline phosphatase activity can be responsible for staining of NADPH-diaphorase activity in certain non-neural cells.

The NADPH-diaphorase (NADPH-d) reaction is frequently used to visualize the diaphorase activity of nitric oxide synthase (NOS). However, this tetrazolium salt procedure can be of limited specificity at sites where non-specific alkaline phosphatase (alP) and NADHd activity co-exist. This is shown in the present paper using methods of catalytic histochemistry for these three enzymes and levamisole as alP inhibitor for certain mouse tissues. In the urothelium, portio, vaginal and endometrial epithelium as well as in some smooth muscle cells alP hydrolyzes NADPH to NADH which in turn serves as substrate for NADHd leading to false-positive formazan production. To exclude this possibility, it is recommended always to include levamisole in the incubation medium if the NADPHd activity of NOS has to be investigated.

Alkaline Phosphatase↗

PubMed1995

Histochemistry of nitric oxide synthase in the nervous system.

Nitric oxide synthase, which generates the physiological messenger molecule nitric oxide, and its associated NADPH diaphorase (NADPHd) activity are distributed throughout selective neuronal populations of the central and peripheral nervous system. Considerable evidence has been accumulated to indicate that NADPHd activity labels cells lacking neuronal nitric oxide synthase, i.e., the specificity of the reaction has to be considered for the reliable detection of the enzyme in neuronal but also non-neuronal tissue. In the present review, critical aspects of nitric oxide synthase visualization in neurones, using its NADPHd activity, are discussed. Furthermore, the organization of the central and peripheral nitric oxide synthase-containing neuronal systems is described. Nitric oxide synthase is present in local cortical and striatal neurones, hypothalamic magnocellular neurones, mesopontine cholinergic neurones, cerebellar interneurones, preganglionic sympathetic and parasympathetic neurones, neurones in parasympathetic autonomic and enteric ganglia and primary viscero-afferent neurones. Finally, injury-related alterations in nitric oxide synthase activity are briefly outlined. In this respect, the histochemistry of nitric oxide synthase may represent a valuable marker for neurochemical, if not structural, alterations observed in neural diseases, regeneration and transplantation.

Animals↗

PubMed1994

Light microscopic visualization of transport ATPase in the chick kidney and intestine using catalytic histochemistry.

To visualize the enzyme Na+/K(+)-ATPase (transport ATPase) in the chick kidney and intestine two recent methods of catalytic histochemistry were modified using capture of inorganic phosphate with lead according to Mayahara et al.(1980) or cerium after Kobayashi et al. (1987). For light microscopy a new step for the visualization of the reaction product was added; lead phosphate was visualized with (NH4)2S and cerium phosphate with the DAB-H2O2-Ni-hexamonium sulfate method. Reaction product was specifically found in the basolateral plasma membrane region of enterocytes and renal epithelial cells (distal tubules, thick ascending limbs of Henle's loops, cortical collecting ducts). Treatment of the sections with 8 mM levamisole and 40 mM L-phenylalanine before and during incubation was necessary to suppress the co-reaction of non-specific alkaline phosphatase in the microvillous zone of proximal tubules and enterocytes. The reaction specificity was controlled with 10 mM ouabain which completely inhibited the basolateral activity in enterocytes and renal epithelial cells. The described methods for transport ATPase are reliable and provide reproducible results in the chick.

Alkaline Phosphatase↗

PubMed1994

When NADPH diaphorase (NADPHd) works in the presence of formaldehyde, the enzyme appears to visualize selectively cells with constitutive nitric oxide synthase (NOS).

The NADPH diaphorase (NADPHd) reaction for nitric oxide synthase (NOS) visualization suffers from the circumstance that the diaphorase activity of NOS represents only part of the total diaphorase activity, and so far all efforts to make the reaction more specific for routine studies failed. The present investigation describes a simple procedure for mouse tissue, which allows the selective staining primarily of neurons, vascular endothelial cells and macula densa cells, those cells where constitutive NOS has been described reliably. In this method unfixed cryosections and 0.5-1% phosphate-buffered formaldehyde containing 0.5 mg NADPH/1 ml and 1 mg nitro BT/1 ml are used. Compared to strong prefixation with formaldehyde after which many additional cells are still positive for NADPHd, presence of formaldehyde in the incubation medium obviously allows the selective reaction of NOS-positive cells. In conclusion, compared with the original technique a more specific method for the visualization of the NADPHd activity of NOS appears to be available now and can be used for NOS studies in all kinds of mammalian species.

Amino Acid Oxidoreductases↗

PubMed1994

Light microscopic visualization of diamine oxidase using a cerium method.

Diamine oxidase (DAOX) can be localized in the light microscope using immunohistochemistry, but a reliable procedure for detecting the activity of the enzyme does not exist. A method was thus developed in which cerium ions trap H2O2, generated by DAOX, and a diaminobenzidine-H2O2-Co sequence serves for the visualization of the primary reaction product cerium perhydroxide. With this method and diamine substrates cadaverine or putrescine, or polyamine substrates spermidine or spermine as substrates, DAOX was localized species-independently but substrate-dependently in the basolateral cytoplasm and/or plasma membrane of small intestinal enterocytes of rats, mice and gerbils. Less, or even no, final reaction product was seen in these cells of guinea-pigs and marmosets. Colonic enterocytes were positive for DAOX solely in gerbils. Furthermore, species-dependent diamine oxidase was present in special cells of the mouse and guinea-pig kidney, immune organs and liver of rats and gerbils, marmoset placenta and in decidual cells of the human, rat and mouse placenta.

Amine Oxidase (Copper-Containing)↗

PubMed1994

Light microscopic visualization of semicarbazide-sensitive amine oxidase (benzylamine oxidase) using a cerium method.

Light microscopic histochemical studies to visualize semicarbazide-sensitive and H2O2-generating amine oxidase (SSAOX, benzylamine oxidase, BAOX; EC 1.4.3.6?) are usually performed with the coupled peroxidatic oxidation technique of Ryder et al. [25]. For methodological reasons this procedure has its limitations and was therefore replaced by a more reliable and easier to perform cerium-DAB-H2O2-Co technique. With this method SSAOX was studied in many organs of various laboratory rodents and marmosets and in human placenta. Independent of the species the enzyme was present mostly in the plasma membrane of nearly all vascular and non-vascular smooth muscle cells. However, there was a species-dependence of SSOX activity; the highest amounts of stain were found in gerbils and marmosets. In addition, the enzyme was found in these two species in the capillary endothelium of some extra-nervous tissues. The plasma membrane localization of SSAOX and plasma membrane-associated H2O2 production suggest a functional role for the enzyme different from that of other amine oxidases which appear to be primarily involved in intracellular amine detoxification or degradation.

Animals↗

PubMed1994

Comparative enzyme histochemistry of the early and term rat decidua with special attention to decidual regression.

As the early rat decidua is believed to fulfil functions other than the late or basal decidua, the question as to whether this difference is reflected in decidual cell metabolism was investigated. Using cryosections of pregnant rat uteri of the 10th, 15th and 21st gestational day, activities of oxyradical-forming enzymes and hydrolases were analysed histochemically. The enzyme activities of decidual stromal cells and fibroblasts of the metrial gland exhibited three main fluctuations. One group of enzyme activities did not change during gestation, a second group decreased or disappeared, and a third group increased or was expressed in the late decidua only. Enzymes of the purine and polyamine pathway, including oxyradical-forming oxidases, were absent from early mesometrial decidual cells, but were highly active in the late regressing decidua and metrial gland. Some acid hydrolases and neutral proteases became active in the mature decidua. The possibility that purine-degrading and oxyradical-forming enzymes support decidual as well as metrial gland regression, and thus placental separation, by direct tissue damage and/or by indirect rupture of lysosomal membranes, inducing the release of acid hydrolases, is considered.

Animals↗

PubMed1993

Light microscopic visualization of monoamine oxidase using a cerium method.

H2O2-generating monoamine oxidase can be visualized in the light microscope with tetrazolium, metal salt (ferricyanide) and coupled peroxidatic oxidation methods. Due to methodological draw-backs these procedures do no allow satisfactory results. In search for an alternative method a light microscopic cerium procedure was designed in which the primary reaction product, cerium perhydroxide, serves for the generation of amplified and intensified diaminobenzidine brown. With this cerium-diaminobenzidine-H2O2-Co method monoamine oxidase was visualized more easily and reliably and with higher sensitivity and more precise localization than with the other techniques. At present this method is considered to be the procedure of choice and was used to re-investigate and investigate the distribution of monoamine oxidase in rats, mice, gerbils, guinea-pigs, marmosets, monkeys and man. In these species many cells and tissues showed monoamine oxidase activity where the enzyme has not yet been found before and the structures with already known monoamine oxidase activity showed an improved localization.

Animals↗