Biochemical properties and cytochemical localization of ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase activity in rat atrial myocytes.

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3. Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine.

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.

Ecto-ATPase activity in cerebellum: implication to the function of synaptic transmission.

The involvement of ATP in synaptic transmission was examined in synapses on granule cells of the rat cerebellum using ecto-ATPase activity. Reaction product was found in a majority but not all synapses between axodendritic, axoaxonic, and dendrodendritic appositions of granule cells and was associated with extracellular surface of both pre- and postsynaptic membranes. Specificity of the detection was justified by using diethyl pyrocarbonate, specific inhibitor of ecto-ATPase activity. These observations provide direct morphological evidence in support of the view that ATP participates in synaptic transmission and indicate functional heterogeneity of synapses in cerebellum.

Detection of ecto-ATPase activity in synaptic plasma membranes for studying extracellular ATP-induced signal transduction.

This paper describes protocol for the correlated light and electron microscopical histochemical visualization of the reaction product of ecto-ATPase activity in brain. The protocol employs a biochemically optimized incubation medium to adjust the appropriate kinetical parameters for detection of the histochemical reaction product by means of confocal laser scanning and electron microscopy. The reaction product is formed when the liberated inorganic phosphate is captured in histochemical reaction by the cerium ions. Confocal microscopy is performed in reflectance mode due to sufficient reflectance properties of the cerium-containing reaction product. Using this procedure, the prominent reaction of ecto-ATPase activity is readily detectable in hippocampus and cerebellum at sites where ATP is supposed to act as a synaptic neurotransmitter: on synapses of neurons in the pyramidal cell layer of hippocampus, in the granule cell layer of the dentate gyrus, and around synapse-containing areas in the granule cell layer of cerebellum. Reaction product is seen in close association with both pre- and postsynaptic membranes and exclusively extracellularly. Specificity of the visualization is justified in control experiments with diethyl pyrocarbonate, specific inhibitor of ecto-ATPase. The procedure is easy to perform, sensitive, and reproducible. It is recommended as a valuable tool in the arsenal of biochemical, immunochemical, and physiological techniques in studying signal transduction induced by extracellular ATP.

Localization of glucose-6-phosphatase activity in the rat cardiac muscle by cobalt-based cytochemistry.

The histocytochemical method for the detection of glucose-6-phosphatase activity has been studied in the rat heart tissue by using cobalt-capture technique. The incubation medium had the following ingredients: cobalt chloride, PIPES-NaOH buffer pH = 6.7, glucose-6-phosphate disodium salt and phenylalanine. Cobalt was revealed by using a solution of 1% ammonium sulfide and was fixed by osmium tetroxide. The most effective concentration of cobalt chloride was 50 mM. With a concentration lower than 30 mM, the reaction products were not determined. With a higher concentration, a small non-specific precipitation was visualized. Ultracytochemically, a strong enzyme reaction was present in the nuclear envelopes of cardiac myocytes, endotheliocytes and in the sarcoplasmic reticulum. These results imply that the method used is valid and can be recommended for studying the localization of glucose-6-phosphatase activity.

Enzyme ultracytochemical demonstration of Ca(++)-ATPase in the rat cerebral cortex.

The localization of Ca(++)-ATPase activity was investigated in the rat cerebral cortex using an ultracytochemical method. Our cytochemical procedure for the detection of enzyme activity is based on an incubation medium consisting of tricine buffer, ATP-disodium salt as substrate, cerium chloride as capturing agent, CaCl2, and levamisole as a nonspecific alkaline phosphatase inhibitor. Final pH was 7.4. Reaction products showing Ca(++)-ATPase activity were localised on the pre- and postsynaptic plasma membrane in association with synaptic vesicles, postsynaptic dendrites and on the axolemma in myelinated nerve fibres. The verification of the main enzymatic properties of Ca(++)-ATPase localization activity is the subject of the following report.

Cytochemical investigation of p-NPPase activity in rat cardiac muscle.

The cytochemical demonstration of the atrial cardiac myocyte pumping activity has been made by detecting p-nitrophenylphosphatase (NPPase), which is used by investigating of Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase activities. The fine ultrastructural localization of these enzymes was studied using cytochemical methods with cerium as a capturing agent. Na(+)-K(+)-ATPase was localized on the atrial muscle cell plasma membrane, T-tubule membrane, endothelial cell nuclear membrane, and cardiac myocyte nuclear membrane. H(+)-K(+)-ATPase was localized on the atrial muscle cells plasma membrane, T-tubule membrane, and sarcoplasmatic reticulum. The present findings indicate that the transporting metabolic activity of the heart as an endocrine organ is realized by the interaction between p-NPPases.

Ultracytochemical characteristic of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (Na(+)-K(+)-ATPase) of rat myocardium.

The ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase within a rat heart has been studied. Our cytochemical procedure for the detection of enzyme activity is based on an incubation medium consisting of tris-maleate buffer, p-nitrophenylphosphate (p-NPP) as a substrate, lead nitrate as a trapping agent, MgCl2, KCl, levamisole, final pH = 7.3. The physiological pH of the incubation medium highly increases the authenticity of the enzymatic reaction and is the main advantage of the technique. Postincubation processing of specimens by 0.1 mol/l tris-maleate buffer pH = 6.0 confirms the specificity of the reaction and removes nonspecific precipitation.

Ultracytochemical demonstration of Ca(++)-ATPase activity in the rat cardiac muscle.

Ca(++)-activated adenosine triphosphatase (Ca(++)-ATPase) was investigated in the rat cardiac muscle at neutral pH using tricine buffer. Reaction products indicating Ca(++)-ATPase activity were localized on the myocardial sarcolemma, sarcoplasmic reticulum, myofilaments, luminal and abluminal surfaces of capillary endothelium plasmatic membrane. The verification of the main enzymatic characteristics of Ca(++)-ATPase localization activity using this cytochemical procedure is under discussion.

Demonstration of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the rat cardiac muscle by cobalt-based cytochemistry.

We have utilized cobalt-reaction technique for histochemical and cytochemical demonstration of ouabain-sensitive, K(+)-dependent p-NPPase (Na(+)-K(+)-ATPase). The incubation medium consisted of: cobalt chloride, tricine buffer, p-nitrophenylphosphate, KCl, and phenylalanine. Final pH 7.4. Ultracytochemically, reaction products were localized along the internal side of sarcolemma and its vesicles, T-tubule membrane, and capillary endothelial cells. These results suggest that the method is reliable and can be used to investigate the localization of Na(+)-K(+)-ATPase activity.

[An electron microscopic study of the localization of the activity of the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase component of the Na, K-ATPase complex in the rat brain]. / Elektronno-mikroskopicheskoe izuchenie lokalizatsii aktivnosti uabain-chuvstvitel'noi, kaliizavisimoi p-nitrofenilfosfataznoi sostavliaiushchei Na, K-ATFaznogo kompleksa golovnogo mozga krysy.

The localization of an ouabain-sensitive potassium-dependent p-nitrophenylphosphatase part of the Na+,K(+)-ATPase complex in the white rat's brain has been studied at the ultrastructural level. The physiological pH of incubation medium highly increases the specificity of ultracytochemical enzyme demonstration. The main characteristics of the enzymatic p-NPP hydrolysis used for this methodological technique are discussed.

[The comparative characteristics of the localization of the activity of alkaline phosphatase and ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase in the myocardium of white rats at the ultrastructural level]. / Sravnitel'naia kharakteristika lokalizatsii aktivnosti shchelochnoi fosfatazy i ouabain-chuvstvitel'noi, kalii-zavisimoi p-nitrofenilfosfatazy v miokarde belykh krys na ul'trastrukturnom urovne.

A comparative characteristic of alkaline phosphatase and Na(+)-K(+)-ATPase localization activity within white rat myocardium is presented at the ultrastructural level. Both different in principle and common features of the enzyme reactional products precipitation are revealed. The original technique is used to determine ouabain-sensitive potassium-dependent p-nitrophenylphosphatase part of Na(+)-K(+)-ATPase complex at physiological pH. The verification of the main characteristics of Na(+)-K(+)-ATPase complex membrane localization activity within the rat myocardium using this cytochemical procedure are discussed.

[Structural changes of the heart in craniocerebral trauma]. / Strukturnye izmeneniia serdtsa pri cherepno-mozgovoi travme.

A study is presented of structural changes in the myocardium due to head injuries. General histological, histochemical, histoenzymological methods and electron microscopy were used. The authors revealed a complex of dystrophic and adaptative changes in the contractile myocardium, interstitial connective tissue and blood circulatory bed. These changes depended directly on the character of the craniocerebral injury and time from the beginning of the trauma.

Ecto-ATPase activity in the rat cardiac muscle: biochemical characteristics and histocytochemical localization.

We used a combined biochemical and histocytochemical approach to study ecto-ATPase in the rat cardiac muscle. The reaction medium employed for histocytochemical detection was optimized in biochemical assays to achieve the highest enzyme activity and lowest inhibition by the capture agent used for visualization of the reaction product. Approximately 70% of the enzyme activity was retained in samples after the fixation procedure. Divalent cations stimulated ecto-ATPase. High activity was detectable within a wide pH range. Histocytochemical reaction was observed at sites at which extracellular ATP can potentially exert its actions on the cardiac muscle: nerve endings, plasma membranes of cardiac myocytes and capillary endothelial cells, and T-tubules. Product of the reaction was found exclusively at the outer surface of the cells. In controls, enzyme activity was abolished by diethyl pyrocarbonate and slightly stimulated by digitonin and concanavalin A, whereas sodium orthovanadate, N-ethylmaleimide, and sodium azide yielded no effect. Our results support the view that cardiac ecto-ATPase is involved in important physiological functions and suggest that its activity may be regulated by the release of ATP from nerve endings.

Dynamics of rat liver ecto-ATPase during development suggests its involvement in bile acid efflux. A cytochemical view.

We studied cytochemical localization of ectoadenosine triphosphatase in the rat liver during development from 15-day-old fetus to 4-week-old and adult animal. First signs of the enzyme activity were found in some of the primitive bile canaliculi of 15-day-old fetuses. The majority of canaliculi, however, did not reveal any reaction product. Although intensity of the cytochemical reaction increased at 20 days of gestation, it still remained relatively low. Intensity of the reaction increased significantly and its product became readily detectable in the liver of newborn rats. Liver of 1-, 2-, and 4-week-old animals showed strong reaction for ecto-ATPase at the luminal surface of the plasma membrane of the bile canaliculi. Liver of adult rats contained a prominent reaction product similar to that seen in newborns, 1-, 2-, and 4-week-old animals. At all stages of fetal development, as well as in postnatal and adult rats, reaction was found only within the hepatic bile canalicular system and exclusively at the luminal surface of the canalicular plasma membrane. Using diethyl pyrocarbonate (DEPC), a specific inhibitor of ecto-ATPase activity, cytochemical reaction was blocked in all examined samples. Results of the present study, taken together with established biochemical and immunological data, provide conclusive morphological evidence in support of the view that canalicular ecto-ATPase is involved in bile acid efflux.

Lipopolysaccharide alters ecto-ATP-diphosphohydrolase and causes relocation of its reaction product in experimental intrahepatic cholestasis.

Lipopolysaccharide (LPS), a bacterial endotoxin, exerts profound inflammatory actions toward various tissues and cells. We induced intrahepatic cholestasis in rats by administration of LPS and followed ecto-ATP-diphosphohydrolase (ecto-apyrase) activity in the liver. The activity of the enzyme had decreased to 77% 2 h after injection compared with the activity in control animals. The maximum decrease was detected 24 h after administration. The activity was found to have partially recovered 1 week after injection, but had yet to reach control levels. In contrast to the decrease in ecto-apyrase activity, there were increases in alkaline phosphatase activity and bilirubin concentration, markers of cholestasis. In response to LPS, the reaction product of ecto-apyrase was found to relocate from the canalicular domain of the plasma membrane of hepatocytes, its predominant localization in the liver of intact animals, to the basolateral and sinusoidal domains. The pattern of histochemical reaction indicated modulation of the enzyme activity and changes in trafficking of intracellular proteins. Taken together, our findings showed that LPS administration alters ecto-apyrase and causes relocation of its reaction product from the canalicular domain of the plasma membrane of hepatocytes in the rat. It is suggested that relocation of the reaction product may be a protective mechanism to enable the hepatocytes to withstand the cytokine-induced metabolic perturbations.

Contralateral thalamic perfusion in patients with reflex sympathetic dystrophy syndrome.

Iodine-123-labelled iodoamphetamine single-photon emission computed tomography of patients with reflex sympathetic dystrophy syndrome showed substantial variation in thalamic perfusion of the side contralateral to the painful limb. The variations are related to time from the onset of symptoms, which suggests that the thalamus undergoes adaptive changes in the course of this neurological disorder.

Intracellular dynamics of alkaline phosphatase-containing granules in electropermeabilized human neutrophils.

Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood. The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon stimulation with AIF4 and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but not with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPgammaS, but not with GDPbetaS, these granules fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization of the alkaline phosphatase-containing granules in cells stimulated with GTPgammaS. The results from this study indicate that GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated with the microtubules in electropermeabilized human neutrophils.

A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils.

In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.

Paradoxical enhancement of spinal-cord-evoked potentials rostral and caudal to the site of progressive cord compression in the cat.

STUDY DESIGN: Analysis of the sequential waveform changes of the spinal-cord-evoked potentials (SCEPs) associated with progressive cord compression in the cat. OBJECTIVES: To document the phenomenon of paradoxical enhancement of SCEPs despite conduction abnormalities and to evaluate its possible significance. SETTING: Kochi Medical School, Kochi, Japan. METHODS: SCEPs were recorded simultaneously at four serial intervertebral levels, from T6-7 to T9-10 caudal to, and at three serial levels from T2-3 to T4-5 rostral to the compression site at T5-6 following epidural stimulation at L6 in 14 cats. RESULTS: Caudal to the compression site, the area of negative peak significantly increased toward maximal values of 277+/-36 (mean+/-SE), 151+/-9 and 110+/-4% as compared to the baseline precompression values (100%) at T6-7, T7-8, and T8-9, respectively. Rostral to the compression site, the area of negative peak significantly increased before subsequent deterioration and reached 105+/-2, 106+/-2, and 104+/-2% at T4-5, T3-4, and T2-3, respectively. The onset of negative peak enhancement, recorded either caudal or rostral to the compression site, showed a close temporal correlation (r>0.8, P&<0.001) with that of the prolongation in latency of SCEPs at T2-3. CONCLUSIONS: A progressive focal conduction block induced by compression of the spinal cord can paradoxically enhance the ascending SCEPs both caudally and, though less consistently, rostrally, representing a warning of the impending risk of paraplegia.