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At least 757 records · Page 42Linked to original sources
PubMed1986

pH sensitivity of the basolateral membrane of the rabbit proximal tubule.

Conventional microelectrodes were used to study the effects of bath pH and bicarbonate concentrations on the basolateral membrane potential (Vbl) of cells from the superficial proximal convoluted (PCT) and proximal straight (PST) tubules of the rabbit kidney perfused in vitro. Bathing solution pH was varied over the range of 5.9-7.4 using either control (22-25 mM) or low bicarbonate (5.0-6.6 mM) Ringer solutions and the appropriate CO2 tensions. The results show a strong pH dependence of the steady-state values of Vbl in both the convoluted and straight tubule segments. The pH-dependent depolarization was approximately 35 mV/pH unit change of the bathing solution in the acid direction and could be demonstrated in CO2-free HEPES-buffered solutions. A depolarizing response to increased bath potassium concentration (HK) was observed that was linearly related to the absolute value of the Vbl under control conditions. Under acidotic conditions, reduced HK depolarizations indicate that a decrease in the relative potassium permeability of the basolateral membrane is the principle mechanism underlying the effects of bath pH on Vbl.

Animals↗
PubMed1986

Active tetraethylammonium uptake across the basolateral membrane of rabbit proximal tubule.

Tetraethylammonium (TEA) uptake was measured in isolated, nonperfused rabbit S2 proximal tubule segments. The TEA cell-to-bath concentration ratio in bicarbonate-Ringer bathing medium was 10-fold higher than that predicted by passive equilibration according to the basolateral electrochemical potential, indicating that TEA uptake is an active process as reported by previous investigators. Removing bicarbonate and CO2 reduced TEA uptake to 22% of control. When bicarbonate-CO2 was replaced by HEPES-O2 or butyrate-O2, TEA uptake was unaltered, but uptake was inhibited when the major buffer anion was omitted. In the presence of 10(-4) M ouabain and bicarbonate-CO2, the TEA cell-to-bath concentration ratio was reduced to 20% of control. TEA uptake in the absence of bicarbonate and CO2 was unaltered by the addition of 10(-4) M ouabain. TEA uptake was inhibited when the bathing medium contained 0 mM K+ or 2 mM Ba2+. These data 1) demonstrate that active basolateral TEA uptake is dependent on medium buffer capacity and 2) support the concept that a portion of TEA uptake occurs via a passive equilibration pathway.

Animals↗
PubMed1996

Effect of alpha-KG in lumen on PAH transport by isolated perfused rabbit renal proximal tubules.

To determine whether dicarboxylate taken up at the luminal membrane could function in the p-aminohippurate (PAH) countertransport at the basolateral membrane, we examined the effect of adding alpha-ketoglutarate (alpha-KG) or glutarate (a nonmetabolized dicarboxylate that is countertransported for PAH at the basolateral membrane) to the luminal perfusate on net secretion of radiolabeled PAH in isolated perfused S2 segments of rabbit proximal tubules. Addition of 100 microM alpha-KG or glutarate to the luminal perfusate in tubules perfused and bathed with HEPES-buffered medium (in the absence of bicarbonate, glycine, lactate, malate, and citrate) produced a reversible twofold stimulation of net PAH transepithelial secretion. Addition of 4 mM LiCl (an inhibitor of Na-dicarboxylate transport that does not directly affect PAH transport) to the luminal perfusate along with alpha-KG eliminated stimulation of net PAH secretion. Addition of 100 microM or 1 mM alpha-KG or glutarate to the luminal perfusate in tubules perfused and bathed with bicarbonate-buffered medium containing glycine, lactate, malate, and citrate had no effect on net PAH transport from bath to lumen. These data indicate that alpha-KG (or glutarate) that enters the tubule cells via the luminal Na-dicarboxylate cotransporter can stimulate net PAH secretion, apparently via countertransport at the basolateral membrane, but only when tubules are not in an optimal metabolic state to produce intracellular alpha-KG.

Animals↗
PubMed1998

Characterization of an intracellular alkaline shift in rat astrocytes triggered by metabotropic glutamate receptors.

The modulation of intracellular pH by activation of metabotropic glutamate receptors was investigated in cultured and acutely dissociated rat astrocytes. One minute superfusion of 100 microM (1S,3R)-1-aminocyclopentane-1, 3-dicarboxcylic acid (ACPD) evoked an alkaline shift of 0.13 +/- 0. 013 (mean +/- SE) and 0.16 +/- 0.03 pH units in cultured (cortical or cerebellar) and acutely dissociated cortical astrocytes, respectively. Alkalinizations were elicited by concentrations of ACPD as low as 1 muM. The ACPD response was mimicked by S-3-hydroxyphenylglycine (3-HPG) and by (s)-4-carboxy-3-hydroxyphenylglycine (4C-3HPG) but was not blocked by alpha-methyl-4-carboxyphenylglycine (MCPG) or (RS)-1-aminoindan-1, 5-dicarboxcylic acid (AIDA), features consistent with an mGluR5 receptor-mediated mechanism. The ACPD-evoked alkaline shift was insensitive to amiloride, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), and the v-type ATPase inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), bafilomycin, and concanamycin. The alkaline response persisted in Na+- or Cl--free saline, but was reversibly blocked in bicarbonate-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions. A bicarbonate-dependent and Na+-independent alkaline shift could also be elicited by either 3 mM caffeine or 1 muM ionomycin. These data suggest that a rise in cytosolic Ca2+ activity is instrumental in triggering the alkalinizing mechanism and that this response is independent of the classic depolarization-induced alkalinization mediated by electrogenic sodium-bicarbonate cotransport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMedDate not supplied

Effect of tooth-bound fluoride on enamel demineralization/ remineralization in vitro.

The effect of tooth-bound fluoride (F) on enamel caries formation was investigated under the condition that loosely bound F was essentially absent. Eighteen thin enamel sections, prepared from the lingual or buccal surfaces of extracted human molars, were embedded in acrylic resin with the enamel surfaces exposed. The sections were placed in a pH 7 remineralizing solution (RS; 1.2 mmol/l Ca, 0.72 mmol/l P, 30 mmol/l KCl, 50 mmol/l HEPES) for 5 days, and were randomly divided into 3 groups: (1) control group that received no treatment, (2) acidulated phosphate fluoride (APF) group that received 5 cycles of a 4 min treatment with APF gel followed by immersion in the RS for 2 days (RS changed daily) and (3) dicalcium phosphate dihydrate (DCPD) - APF group that received 5 cycles of a 4-min pH 2.1 DCPD-forming solution followed by 4 min APF gel and then placed in the RS for 2 days. After the treatment cycles, the sections were washed in a constant composition F titration system to remove loosely bound F. An in vitro model, which consisted of cycles of de- (6 h) and remineralization (18 h) each day for 5 days, was used to produce caries-like lesions in the specimens. The DeltaZ (mineral loss) values, measured by quantitative microradiography, of the lesions formed in the three groups were (mean +/- standard deviation; n = 6) 91.2+/-12.3 microm for the control group, 41.3+/-10.1 microm for the APF group and 21.2+/-4.8 microm for the DCPD-APF group. The same system produced lesions in untreated shark enamel with a mean DeltaZ of 4.4+/-0.3 microm (n = 12). One-way fixed-effects ANOVA indicated that mineral loss was significantly different among the different groups (p<0.05). The results showed that enamel resistance to lesion formation increased with increasing tooth-bound F content. Shark enamel was much more resistant to demineralization than human enamel.

Acidulated Phosphate Fluoride↗
PubMed1996

Effect of sodium hypochlorite treatment on remineralization of human root dentine in vitro.

Dentine consists simplified of mineral and of several organic components. Sodium hypochlorite (NaOCl) is a well-known nonspecific proteolytic agent capable to remove organic material. The aim of this study was to investigate the influence of organic material removal from artificial dentine lesions by means of NaOCl pretreatment on subsequent remineralization with and without fluoride. Human root dentine samples were demineralized in an acidic gel (pH = 5) at 37 degrees C for 2 weeks. After 2 min of pretreatment with a 0.4, 2 or 10% NaOCl solution, the samples were remineralized in a 20 mM HEPES buffer (pH = 7) containing 1.5 mM Ca(2) and 0.9 mM phosphate with or without addition of 10 ppm F(-) as NaF at 37 degrees C for 8 days. Mineral profiles were assessed by means of transversal microradiography after diol treatment to avoid shrinkage caused by drying. In a separate experiments the dentine contraction caused by 10% NaOCl was assessed. The contraction (negligible for sound dentine) was found to be about 12% for the lesions. The remineralization results showed that pretreatment with a 10% NaOCl solution for 2 min, increased lesion remineralization. After NaOCl treatment, the amount of accumulated mineral increased by about 27% without F in the remineralization solution, and by about 4% with 10 ppm in solution. The in vitro results suggest that removal of organic materials from dentine lesions is an interesting approach to enhance remineralization.

Adolescent↗
PubMed1991

Impurity in buffer substances mimics the effects of ATP on soluble 5'-nucleotidase.

An impurity, probably an anion, present in some batches of the buffer substances 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), 2-morpholinoethane sulfonic acid (Mes) and piperazine-1,4-bis(2-ethane sulfonic acid (Pipes), activates the soluble 5'-nucleotidase from rat kidney. The affinity of the enzyme for 5'-IMP and the Vmax were both increased by the unidentified activator. ATP and 2,3-diphosphoglycerate, known activators of the soluble 5'-nucleotidase, had no effect if the incubation media were buffered with batches containing high concentrations of the activating impurity. These results suggest that the impurity interacts with the soluble 5'-nucleotidase at the same site as ATP and 2,3-diphosphoglycerate, however with a much higher affinity than these two compounds. It is possible that the same impurity might interfere with other proteins for which ATP is a substrate or a ligand.

5'-Nucleotidase↗
PubMed1992

Effects of 2,4-dinitrophenol or low [ATP]i on cell excitability and action potential propagation in guinea pig ventricular myocytes.

Inhibition of aerobic metabolism leads to a major disruption of cardiac cell homeostasis. The purpose of the present study was twofold: 1) We determined the relative importance of junctional and nonjunctional membrane resistance (Rj and Rm, respectively) in the development of propagation failure during inhibition of aerobic metabolism in guinea pig ventricular cell pairs. 2) We used the patch-action potential clamp technique in single ventricular myocytes to study some of the properties of the membrane channels that are responsible for shortening of action potential duration and eventual failure of cell excitation after metabolic blockade. In most experiments, whole-cell patch pipettes were filled with a solution containing 1 mM EGTA, 5 mM HEPES, and 5 mM ATP. Our results in cell pairs showed that pharmacological inhibition of aerobic metabolism with the mitochondrial uncoupler 2,4-dinitrophenol (DNP) led to a drop in Rm followed by an increase in Rj. The increase in Rj was not sufficient to cause a measurable delay in cell-to-cell propagation, whereas the drop in Rm consistently led to failure of cell excitation. Similar results were obtained in additional experiments in which the EGTA concentration in the pipette was reduced to 50 microM. Similar results were also obtained by loading the recording patch pipettes with a solution containing only 0.1 mM ATP. Our patch-action potential clamp experiments, on the other hand, revealed that DNP induced the opening of time- and voltage-independent membrane channels, with a unitary conductance of 23 pS. The channels allowed for the passage of outward current in the voltage range of the action potential, and the increase in membrane patch conductance correlated with the observed shortening of action potential duration during DNP superfusion. Our experiments provide the first simultaneous recordings of action potentials and DNP-induced channel currents in guinea pig ventricular myocytes. Overall, the data provide new evidence for the understanding of the cellular and subcellular mechanisms involved in the development of slow conduction velocity and propagation block after metabolic blockade.

2,4-Dinitrophenol↗
PubMed1988

Glial swelling during extracellular acidosis in vitro.

Intracellular and extracellular acidosis may determine the ultimate outcome for brain tissue in cerebral ischemia. An extracellular acidosis that occurs in the penumbra zone was investigated in vitro as to its role in the formation of cytotoxic cell swelling. For that purpose, C6 glioma cells or primary cultured astrocytes were suspended in normal isotonic medium in normoxia during acidification to a final pH of 6.2. The cell volume response was determined by flow cytometry using hydrodynamic focusing, which allows one to recognize changes in cell size of less than 1%. A threshold pH of 6.8 was found that had to be crossed to induce cell swelling by acidosis. Once pH fell below this threshold, the increase in cell size appeared to be an all-or-nothing phenomenon. The cells rapidly assumed a final cell size of 115% of normal in the case of C6 glioma or of 118% in the case of primary cultured astrocytes independent of the actual level of acidosis or the duration of exposure. Acidosis-induced glial swelling could be significantly attenuated by 1) addition of amiloride, 2) administration of acetazolamide, or 3) replacement of bicarbonate buffer against N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Replacement of extracellular Na+ by choline chloride led to complete prevention of the acidosis-induced cell swelling. Taken together, the findings strongly indicate a central involvement of Na+/H+ and Cl-/HCO3- exchange mechanisms in the development of cell swelling under these conditions. Activation of the Na+/H+ antiporter can be considered an attempt to maintain a normal intracellular pH at the expense of an abnormal cell volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed2003

HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry.

In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPES-glutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.

Buffers↗
PubMed1991

The standard Romanowsky-Giemsa stain in histology.

A new and technically simple Romanowsky-Giemsa (RG) stain is proposed as a standardized technique for use in histology. An RG stock solution (pure azure B 7.5 g/l, eosin Y as eosinic acid 1.2 g/l in dimethylsulfoxide) is diluted to form the working solution with HEPES-buffer, pH 6. Staining time is 30-90 min after formol-calcium solution (or 2-4 hr after formaldehyde-organic acid mixtures). The resulting overstained sections are to be differentiated. A tannic acid-acetic acid combination in an isopropanol-water mixture was found to give optimum results within 100 sec. Subsequent dehydration is in isopropanol only. The staining pattern obtained is polychrome. The distribution of colors in detail is influenced by the modes of pre- and posttreatment. Of practical interest is the development of green and greenish blue colors on collagen fibrils which contrast strongly against the pink of sarcoplasm. For this and other reasons, this RG stain version seems suitable to replace the trichrome Gomori-type trichrome stains under appropriate processing conditions.

Animals↗
PubMed1991

Vanadyl causes hydroxyl radical mediated degradation of deoxyribose.

Vanadyl caused a time- and dose-dependent degradation of deoxyribose to carbonyl products detectable with thiobarbituric acid. This process was inhibited by catalase, ethanol or HEPES; whereas superoxide dismutase was without effect. Vanadate did not substitute for vanadyl even in the presence of a source of O2- plus H2O2; but it did so in the presence of reductants such as thiols or NADH. It appears that hydrogen peroxide, generated by the autoxidation of vanadyl, is reduced by vanadyl to the hydroxyl radical; which, in turn, was responsible for the degradation of deoxyribose. A similar process might contribute to the toxic and pharmacological effects of vanadium salts.

Catalase↗
PubMed2002

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro.

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.

Acetylcholine↗
PubMedDate not supplied

Cryopreservation of a small number of fresh human testicular spermatozoa and testicular spermatozoa cultured in vitro for 3 days in an empty zona pellucida.

It is known that the motility of human testicular sperm can be improved when they are cultured in vitro for a few days. The purpose of this study was to determine whether it is better to freeze human testicular spermatozoa on the day of biopsy (fresh) or after they were cultured for 3 days. A modified, single-sperm freezing technique was used in this study. The study consisted of two parts: (1) ejaculated spermatozoa were used to examine the influence of different concentrations of glycerol and synthetic serum substitute (SSS) on the survival rate after cryopreservation, and (2) the survival rates between cryopreserved fresh testicular spermatozoa (Group 1) and testicular spermatozoa that were cultured for 3 days before freezing (Group 2) were compared. Empty zonae pellucidae were obtained from mouse eggs. Five to 10 motile spermatozoa were selected and injected into an empty zona pellucida. For freezing, the zona pellucida with spermatozoa was transferred into a HEPES-buffered human tubal fluid containing different concentrations of glycerol and kept at room temperature for 10 to 15 minutes, and then loaded into a 0.25-ml-plastic straw. The straws were exposed to liquid nitrogen vapor for 2 hours and then plunged into liquid nitrogen. For thawing, the straws were taken out of liquid nitrogen and placed into a 37 degrees C waterbath for 25 to 30 seconds. There was no statistically significant difference in survival rates between 3% and 10% SSS with different glycerol concentrations. There was no statistically significant difference in the survival rates of spermatozoa between Group 1 and Group 2 after cryopreservation. It appears that in vitro culture of testicular spermatozoa before freezing does not increase survival rate.

Animals↗
PubMed1992

Evidence for the participation of proteinases released by Candida albicans in the early killing of peritoneal macrophages in vitro.

1. We have investigated the possibility that proteinases released by Candida albicans participate in the early killing of three types of mice peritoneal macrophage (resident, thioglycollate-elicited, or Con A-activated) in vitro. 2. Phagocytic assays were performed by incubation of macrophages and C. albicans together at a 1:10 ratio for 30 min at 37 degrees C in RPMI medium buffered to pH 7.0 with 12 mM Hepes without serum. With no albumin added to the medium, the macrophages were 85% to 100% damaged and unviable, to a greater extent than expected from the proportion of phagocytic cells containing germ tubes. When 10 mg/ml of albumin was added to the medium, however, 90% of the macrophages remained viable for the 2 hours of the phagocytic assay, suggesting that albumin may have acted as a substrate for or inhibitor of proteinases released by C. albicans, thereby protecting the macrophage from the proteolytic action of the proteinases. 3. The phagocytosis of IgG-coated erythrocytes was reduced to 43% when IgG was preincubated with the supernatant from C. albicans cultures, but the addition of 10 mg/ml albumin or of 5 micrograms/ml pepstatin (an inhibitor of C. albicans acid proteinases) to the same supernatants prevented the effect on phagocytosis of IgG-coated erythrocytes. 4. These results suggest that proteinases released from C. albicans are involved in the early killing of macrophages.

Albumins↗
PubMed2004

Biochemical relevance between oxidative/carbonyl stress and elevated viscosity of erythrocyte suspensions.

The effects of various aldehydes such as secondary products of peroxidized lipids and other aldehydes on rheological parameters and their relation with aging process were studied. Malondialdehyde (MDA) in different concentrations can increase significantly viscosity and plastic viscosity of erythrocytes suspended in plasma and HEPES buffer solution. Simultaneously, oxidation induced by Fe2+ can also enhance the viscosity of erythrocyte suspensions. All of these data suggest that MDA as one of the most studied unsaturated carbonyl products of lipid peroxidation leading to protein crosslinking may carry important information in understanding carbonyl stress-related rheological change in acute as well as chronic diseases and aging.

Blood Viscosity↗
PubMedDate not supplied

HOPE--a novel tool for the pathologist.

The growing demand for the detection of prognostic and diagnostic biomarkers by application of molecular-biological techniques has been slowed down to a large degree by the use of formalin and its influence onto antigenic structures and nucleic acids, although other reasons also exist. With regard to morphology a limitation in the possibilities, compared to frozen sections, takes place. With the introduction of the HOPE (Hepes glutamic acid buffer mediated Organic solvent Protection Effect) -fixation to date both have become possible; substantially enlarged possibilities concerning the detection of DNA, RNA and proteins in combination with excellent morphological results comparable to formalin-fixed tissues. We also highlight some aspects from our lung specific work during the last years. Immunohistochemical markers for differential diagnostics of malignancies in the lungs are discussed; their enhanced detectability using the HOPE-technique is pathbreaking for the future. The same holds true for the substantially enhanced detectability of germs (e.g. mycobacteria) in HOPE-fixed tissues.

Buffers↗
PubMed1991

Spontaneous and pharmacologically-induced vasoconstrictive responses of rat aortic rings are attenuated by balloon angioplasty.

The effects of arterial dilatation with differently sized PTCA catheters on prestretch-induced, receptor-induced and voltage-induced vasoconstriction was investigated. Rat aortic rings were kept incubated in HEPES buffer for 0,3,6, and 12 hours before mounting in the experimental set-up for contraction measurements. Balloon dilation (BD) was applied prior to incubation or directly before measurement following a period of incubation. Isometric force was measured after applying a prestretch to the vascular rings. The ring then contracted spontaneously or, if not, was brought to contraction by norepinephrine (NE, 50 microM) or potassium ions (K+, 30 mM). Prestretch-induced vasoconstriction reached maximum values after 6 hours of incubation. NE-induced vasoconstriction was maximal at 6 hours of incubation and K(+)-induced vasoconstriction kept rising up to 12 hours of incubation. The addition of enoximone resulted in vasodilatation (ED50 = 0.1 microM) of prestretch-induced vasoconstriction, but was less potent (ED50 = 168 microM) if added to NE-induced or K(+)-induced vasoconstricted rings. BD applied before the incubation period reduced prestretch-induced, receptor-induced and voltage-induced contractions (the effectivity decreasing in that order) and did not elicit vasospastic activity. BD applied after the incubation period and immediately before the contraction measurements prevented the occurrence of prestretch-induced spontaneous contractions, caused a nonsignificant (p = 0.08) decrease of NE-induced vasoconstriction, and had no effect on K(+)-induced vasoconstriction. It is concluded that this model to study spontaneous and induced vasoactivity elucidates the consequences of the application of balloon dilatation in nonatherosclerotic vascular rings.

Angioplasty, Balloon↗