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PubMed2002

The heart of Ciona intestinalis: eicosanoid-generating capacity and the effects of precursor fatty acids and eicosanoids on heart rate.

Eicosanoids are a group of oxygenated fatty-acid derivatives formed from C20 polyunsaturated fatty acids including arachidonic and eicosapentaenoic acids. In mammals, these compounds have been shown to be key molecules in several physiological processes including regulation of the vascular system. This study determined whether eicosanoids or their precursors are involved in the regulation of heart rate in the sea squirt Ciona intestinalis. Eicosanoid generation by both heart and blood cells was measured. The major lipoxygenase products formed were both derivatives of eicosapentaenoic acid, namely 8- and 12-hydroxyeicosapentaenoic acids (8-HEPE and 12-HEPE). Smaller amounts of 8,15-dihydroxyeicosapentaenoic acid (8,15-diHEPE) were also formed. The cyclo-oxygenase product prostaglandin E was also found in small amounts in the heart. Isolated hearts were exposed either to these fatty acid precursors or to 8-HEPE, 12-HEPE or prostaglandin E3, and the effect on heart rate was recorded. Both eicosapentaenoic and arachidonic acids stimulated the heart rate at concentrations between 50 and 200 micromol l(-1). 12-HEPE (5 micromol l(-1)) and prostaglandin E3 (50 micromoll(-1)) caused a modest increase in heart rate, while 8-HEPE had no significant effects at any of the time periods studied (<or=180 min). Overall, the results show that arachidonic and eicosapentaenoic acids have limited effects on heart rate and only at concentrations unlikely to be routinely liberated in vivo. Similarly, the eicosanoids tested had a minor stimulatory activity on heart rate. The potential mechanisms for this stimulation are discussed. Overall, these results suggest that such compounds are of limited importance in regulating the heart and vascular system of sea squirts.

Animals↗
PubMed1989

Effect of deproteinization and reagent buffer on the enzymatic assay of L-carnitine in serum.

Tris and HEPES were systematically compared as buffers for the enzymatic assay of L-carnitine. The deproteinization methods preceding the assay were also compared. The following conclusions were drawn. 1. Both Tris and HEPES act on the catalytic site of the enzyme, acetylCoA: carnitine O-acetyltransferase (EC 2.3.1.7), which is used for the conversion of L-carnitine to acetylcarnitine. HEPES is a competitive inhibitor, and no acetylated product of HEPES is formed. In the presence of Tris a limited amount of acetylTris is formed, and an appropriate blank corrects for this effect. 2. The incubation time of the assay is strongly influenced by the preceding deproteinization method. The enzyme is influenced by inorganic salt, which acts as a competitive inhibitor. 3. If Tris is used in place of HEPES in end-point assays, optimal conditions and shorter assay times are achieved with less enzyme and less acetylCoA, provided more elaborate deproteinization methods are used. 4. The HEPES system is more costly, but preferable for the determination of both total and free L-carnitine in combination with a matched deproteinization method.

Buffers↗
PubMed2002

Intracellular pH plays a critical role in glucose-induced time-dependent potentiation of insulin release in rat islets.

The underlying mechanisms of glucose-induced time-dependent potentiation in the pancreatic beta-cell are unknown. It had been widely accepted that extracellular Ca(2+) is essential for this process. However, we consistently observed glucose-induced priming under stringent Ca(2+)-free conditions, provided that the experiment was conducted in a HEPES-buffered medium as opposed to the bicarbonate (HCO(3)(-))-buffered medium used in previous studies. The critical difference between these two buffering systems is that islets maintain a lower intracellular pH in the presence of HEPES. The addition of HEPES to a HCO(3)(-)-buffered medium produced a dramatic decrease in the intracellular pH. If it is the lower intracellular pH in islets in a HEPES-buffered medium that is permissive for glucose-induced time-dependent potentiation (TDP), then experimental lowering of intracellular pH by other means should allow TDP to occur in a Ca(2+)-free HCO(3)(-)-buffered medium, where TDP normally does not occur. As expected, experimental acidification produced by dimethyl amiloride (DMA) allowed glucose to induce TDP in a Ca(2+)-free HCO(3)(-)-buffered medium. DMA also enhanced the priming normally present in HEPES-buffered media. Priming was also enhanced by transient acidification caused by acetate. Experimental alkalinization inhibited the development of priming. In the presence of Ca(2+), the magnitude of glucose-induced TDP was higher in a HEPES-buffered medium than in an HCO(3)(-)-buffered medium. In summary, glucose-induced priming was consistently observed under conditions of low intracellular pH and was inhibited with increasing intracellular pH, irrespective of the presence of extracellular Ca(2+). These data indicate that glucose-induced TDP is critically dependent on intracellular pH.

Animals↗
PubMed1990

Platelet-neutrophil interactions. 12S,20- and 5S,12S-dihydroxyeicosapentaenoic acids: two novel neutrophil metabolites from platelet-derived 12S-hydroxyeicosapentaenoic acid.

Dietary marine n-3 polyunsaturated fatty acids have demonstrated an antiinflammatory potential in epidemiologic and intervention studies in humans. Proposed mechanisms, involving only leukocytes, fall short of explaining this potential completely. Enriched by dietary means with eicosapentaenoic acid (EPA), stimulated human platelets release substantial amounts of eicosapentaenoic acid and 12S-hydroxyeicosapentaenoic acid (12S-HEPE) in addition to 12S-hydroxyeicosatetraenoic acid (12S-HETE) derived from arachidonic acid. Human neutrophils metabolize 12S-HETE to 5S,12S-DiHETE when stimulated, whereas unstimulated neutrophils produce 12S,20-DiHETE. This study was undertaken to characterize metabolism of 12S-HEPE in human neutrophils. We demonstrate herein for the first time that 12S-HEPE is metabolized by human neutrophils. In unstimulated neutrophils 20-hydroxylation to 12S,20-DiHEPE occurs, whereas in stimulated neurtrophils 5-lipoxygenation to 5S,12S-DiHEPE takes place. The structures of these metabolites were characterized by their relative retention times on reversed-phase high pressure liquid chromatography, by their UV absorbance spectra, and by gas-liquid chromatography-mass spectrometry. With increasing amounts of 12S-HEPE, stimulated neutrophils produced increasing amounts of 5S,12S-DiHEPE, which is virtually inactive biologically. Concomitantly, production of the potent chemokinetic and chemoattractant arachidonic acid derivative leukotriene B4 decreased. Thus, 12S-HEPE can compete with endogenous arachidonic acid for 5-lipoxygenation in stimulated human neutrophils. 12,20-DiHEPE, LTB5, and 5S,12S-DiHEPE were detectable after coincubating EPA-enriched platelets with unenriched neutrophils, and arachidonic acid-derived 5-lipoxygenase products were decreased. We conclude that 12S-HEPE can participate in platelet-neutrophil interactions in a manner similar to 12S-HETE. By providing competing substrates for neutrophil 5-lipoxygenase, platelets might contribute to the antiinflammatory potential of dietary n-3 fatty acids through platelet-neutrophil interaction.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMedDate not supplied

Novel role of extracellular carbon dioxide in lymphocyte proliferation in culture.

CO(2)/HCO(3)(-) buffering system is indispensable to maintain the pH of culture media for long-term cell culture. Now-a-days, the zwiterionic hydrogen buffer HEPES is widely used as an additional buffer in the commonly used culture media. There are reports on the successful use of HEPES-buffered media, under CO(2)/HCO(3)(-) free conditions, for long-term cell cultures. However, still CO(2)/HCO(3)(-) buffering system is widely used. We aimed at investigating the reason for this. We found that lymphocytes proliferate in response to concanavalin A only in HCO(3)(-)-buffered medium in the presence of 5% CO(2), but not in the HEPES-buffered medium in the absence of CO(2). However, lymphocyte proliferation was observed in HEPES-buffered medium in the presence of 5% CO(2) and in the absence of HCO(3)(-). On the other hand, a low level proliferation was observed in HEPES-buffered medium supplemented with HCO(3)(-) in the absence of CO(2). Supplementation of the culture medium with TCA cycle intermediates and the precursors for the salvage pathway of nucleotide synthesis did not support the lymphocyte proliferation at all. Based on these findings and other reports, we suggest that extracellular CO(2) plays a novel role in cell proliferation.

Animals↗
PubMed1988

Radicals from "Good's" buffers.

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.

Buffers↗
PubMed2002

Astrocyte stellation in saline media lacking bicarbonate: possible relation to intracellular pH and tyrosine phosphorylation.

Primary cultures of astrocytes exhibit a polygonal morphology, but on treatment with agents that increase cAMP they change to stellate cells. We found that astrocyte stellation also occurred on replacing the culture medium with saline buffered with HEPES. However, stellation did not occur when the medium was replaced with saline buffered with bicarbonate/CO(2) provided Ca(2+) was present. Since exposure of astrocytes to media lacking bicarbonate results in a decrease in intracellular pH (pH(i)) we sought evidence for an association between pH(i) and morphology. Astrocytic pH(i) was monitored for 60 min after transferring the cells to HEPES or bicarbonate-buffered saline. HEPES-induced stellation was associated with transient acidification which coincided with the morphological changes. Acidification was not observed in cells transferred to bicarbonate-saline. However when cytoplasmic acidification of cells in bicarbonate-saline was induced pharmacologically, rapid stellation occurred. Stellation induced by cAMP is reversed by activation of the RhoA pathway with lysophosphatidic acid (LPA). Here we found that LPA inhibited HEPES-induced stellation, but only with Ca(2+) present. Inhibition of stellation by LPA+Ca(2+) was associated with transient acidification followed by modest alkanization. A close association of tyrosine phosphorylation with stellation and pH(i) was observed. Thus incubation of astrocytes in HEPES-saline with orthovanadate to inhibit dephosphorylation abolished stellation and acidification; conversely incubation of cells in bicarbonate-saline with genistein to inhibit tyrosine kinases caused stellation and major acidification. Acidification may be one of several factors resulting in stellation, but it is not a necessary factor since stellation without acidification was observed in bicarbonate-saline lacking Ca(2+).

Ammonium Chloride↗
PubMed2002

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration.

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The Km value obtained from iron uptake kinetics was 4.5 microM, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.

Animals↗
PubMed1982

Metabolism of Babesia parasites in vitro. Comparison of several zwitterion buffers for the in vitro incubation of Babesia rodhaini.

The effect on the haemoprotozoan parasite Babesia rodhaini of adding a zwitterion buffer, HEPES, to a basal medium was studied by comparing the glucose uptake, ATP concentration, medium pH and infectivity of parasitized rat erythrocytes every 4 h during 24 h incubation in either Krebs Ringer phosphate (KRP) or KRP plus 28 mM HEPES. KRP plus HEPES was found to be superior to KRP for the maintenance of glucose uptake, erythrocyte ATP concentration, medium pH and infectivity of the parasite. Having established that HEPES improved the basal KRP medium, HEPES was then compared to three other buffers TES, BES and TRIS, to determine their relative effects on B. rodhaini during 18 h of incubation. At 28 and 56 mM concentration there was no significant difference in glucose uptake or lactate production among the 4 buffers. However, TES maintained the medium pH best, and BES preserved the infectivity of the parasite best. Further comparison of TES and BES at 10, 20, 30 and 40 mM established 50 mM TES to be best for buffering the pH of the medium and maintaining infectivity of B. rodhaini.

Adenosine Triphosphate↗
PubMed2003

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments.

NO functions ubiquitously as a biological messenger but has also been implicated in various pathologies, a role supported by many reports that exogenous or endogenous NO can kill cells in tissue culture. In the course of experiments aimed at examining the toxicity of exogenous NO towards cultured cells, we found that most of the NO delivered using a NONOate (diazeniumdiolate) donor was removed by reaction with the tissue-culture medium. Two NO-consuming ingredients were identified: Hepes buffer and, under laboratory lighting, the vitamin riboflavin. In each case, the loss of NO was reversed by the addition of superoxide dismutase. The effect of Hepes was observed over a range of NONOate concentrations (producing up to 1 microM NO). Furthermore, from measurements of soluble guanylate cyclase activity, Hepes-dependent NO consumption remained significant at the low nanomolar NO concentrations relevant to physiological NO signalling. The combination of Hepes and riboflavin (in the light) acted synergistically to the extent that, instead of a steady-state concentration of about 1 microM being generated, NO was undetectable (<10 nM). Again, the consumption could be inhibited by superoxide dismutase. A scheme is proposed whereby a "vicious cycle" of superoxide radical (O(2)(.-)) formation occurs as a result of oxidation of Hepes to its radical species, fuelled by the subsequent reaction of O(2)(.-) with NO to form peroxynitrite (ONOO(-)). The inadvertent production of ONOO(-) and other reactive species in biological media, or the associated loss of NO, may contribute to the adverse effects, or otherwise, of NO in vitro.

Animals↗
PubMed1999

Clearance of large Ca2+ loads in a single smooth muscle cell: examination of the role of mitochondrial Ca2+ uptake and intracellular pH.

Redistribution of cytosolic free Ca2+ following Ca2+ influx into the cytoplasm was studied in single smooth muscle cells isolated from guinea-pig urinary bladder. Voltage-clamped cells were loaded with a low-affinity fluorophore Indo-1FF. A decay of free intracellular Ca2+ ([Ca2+]i) after the termination of the depolarizing pulse (1 s from -50 mV to +20 mV) was fitted with a single exponential and the effect of various substances on the time constant was compared. At a holding potential of +80 mV the [Ca2+]i decay was 1.56 times slower compared to that at -50 mV suggesting the presence of a voltage-dependent process redistributing Ca2+. In the presence of cyclopiazonic acid (CPA, 10 microM), an inhibitor of sarco(endo)plasmatic Ca2+ pump (SERCa), the [Ca2+]i decay was 3.93 times slower than that in the absence of the inhibitor. Introduction of a polycation Ruthenium Red (RR) (20 microM), an inhibitor of the mitochondrial Ca2+ uniporter, into a cell or collapsing a transmitochondrial H+ gradient with the protonophore CCCP (2 microM) slowed down the [Ca2+]i decay 6.05-fold and 9.78-fold, respectively. The apparent amplitude of [Ca2+]i increments was also increased by CCCP. Increasing H+ buffering power in the intracellular solution from 10 mM to 40 mM of HEPES greatly reduced the effect of CCCP on [Ca2+]i decay. A further increase in HEPES concentration to 100 mM eliminated the effects of CCCP both on the time course of [Ca2+]i decay and on the amplitude of [Ca2+]i increment. Perfusion of RR together with 100 mM HEPES into the cytoplasm was without effect on the decay time course of [Ca2+]i. The effect of CPA on [Ca2+]i decay was also reduced in cells loaded with 100 mM HEPES; the time constant in the presence of CPA was slowed down by a factor of 2.18. Application of 10 mM Na(+)-butyrate to the cells loaded with 10 mM HEPES resulted in a slowing down of [Ca2+]i decay: the time constant was increased by a factor of 5.84. Measurement of intracellular pH with SNARF-1 confirmed cytoplasmic acidification during application of Na(+)-butyrate and CCCP. It is concluded that the contribution of mitochondrial Ca2+ uptake to the rapid [Ca2+]i decay is much less than could be extrapolated from action of protonophores in these smooth muscle cells. The results also demonstrate the importance of intracellular pH for Ca2+ handling in the cytoplasm of smooth muscle cells.

Animals↗
PubMed1990

Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2.

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.

Adrenal Medulla↗
PubMed2001

Initial effect of sodium bicarbonate on intracellular pH depends on the extracellular nonbicarbonate buffering capacity.

OBJECTIVE: The effect of sodium bicarbonate on intracellular pH under conditions close to those in vivo, with both bicarbonate and nonbicarbonate buffering systems, is unknown. We postulated that this effect depends on the nonbicarbonate buffering capacity because the alkali-induced back-titration of these buffers results in a concentration-dependent release of CO2 in the extracellular space, leading to a decrease in intracellular pH. DESIGN: The study was conducted in two stages. First, human hepatocytes were perfused with pH 7 bicarbonate-buffered medium (5 mM HCO3-, 20 torr Pco2) containing no nonbicarbonate buffer or small amounts (5 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES]) or large amounts (20 mM HEPES) of nonbicarbonate buffer. Second, the changes in intracellular pH of hepatocytes placed in acidotic human blood (pH 7, 5 mM HCO3-, 20 torr Pco2) at three hematocrits (40%, 20%, and 5%) were measured. SETTING: Research laboratory at a medical university. SUBJECTS: Cryopreserved human hepatocytes thawed the day before the experiments. INTERVENTIONS: Sodium bicarbonate was infused for 10 mins to increase the HCO3- concentration from 5 to 30 mM. In the second part, 20 mM sodium bicarbonate was added directly to the blood bathing the cells. MEASUREMENTS AND MAIN RESULTS: The intracellular pH was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein in its esterified form, acetoxy-methyl ester, by using a single-cell imaging technique. Gas analyses were performed before and during the sodium bicarbonate load. Sodium bicarbonate caused a decrease in intracellular pH with all media except the artificial medium containing no HEPES. This decrease was small in media with low nonbicarbonate buffering capacity (5 mM HEPES and 5% hematocrit blood) and large in media with high nonbicarbonate buffering capacity (20 mM HEPES and 40% hematocrit blood). The change in intracellular pH was linked closely to the change in Pco2 caused by the sodium bicarbonate. CONCLUSIONS: The effect of sodium bicarbonate on intracellular pH depends on changes in Pco2 in the medium bathing the cells. The increase in Pco2 is correlated with the extracellular nonbicarbonate buffering capacity because of the release of H+ ions coming from the back-titration of these buffers. We conclude that sodium bicarbonate may exacerbate cell acidosis under buffering conditions close to those in vivo and that the initial changes in cell pH caused by sodium bicarbonate depend on the extracellular nonbicarbonate buffering capacity.

Blood↗
PubMed2003

Contractile reserve but not tension is reduced in monocrotaline-induced right ventricular hypertrophy.

The objective of this study was to evaluate the role of right ventricular hypertrophy on developed tension (F(dev)) and contractile reserve of rat papillary muscle by using a model of monocrotaline (Mct)-induced pulmonary hypertension. Calcium handling and the influence of bicarbonate (HCO(3)(-)) were also addressed with the use of two different buffers (HCO(3)(-) and HEPES). Wistar rats were injected with either Mct (40 mg/kg sc) or vehicle control (Con). Isometrically contracting right ventricular papillary muscles were studied at 80% of the length of maximal developed force. Contractile reserve (1 - F(dev)/F(max)) was calculated from F(dev) and maximal tension (F(max)). Calcium recirculation was determined with postextrasystolic potentiation. Both groups of muscles were superfused with either HCO(3)(-) (Con-B and Mct-B, both n = 6) or HEPES (Con-H and Mct-H, both n = 6) buffer. With hypertrophy, contractions were slower but F(dev) was not changed. However, F(max) was decreased (P < 0.05). With HCO(3)(-), F(max) decreased from 23.8 +/- 6.5 mN.mm(-2) in Con-B, to 13.7 +/- 3.3 mN.mm(-2) in Mct-B. With HEPES, it decreased from 16.3 +/- 3.5 mN.mm(-2) (n = 6, Con-H) to 8.3 +/- 1.6 mN.mm(-2) (Mct-H). Contractile reserve during hypertrophy was therefore also decreased (P < 0.05). With HCO(3)(-), it decreased from 0.73 +/- 0.03 (Con-B) to 0.55 +/- 0.04 (Mct-B). With HEPES, it decreased (P < 0.001) from 0.64 +/- 0.07 (Con-H) to 0.19 +/- 0.06 (Mct-H). The recirculation fraction decreased (P < 0.05) from 0.59 +/- 0.04 in Con-B to 0.44 +/- 0.04 in Mct-B. We conclude that contractile reserve and recirculation fraction are impaired during hypertrophy, with a stronger effect under HEPES than HCO(3)(-) superfusion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

[Aspirated sperm activation from deferens in an ionic media similar to seminal plasma].

OBJECTIVE: To compare motility on sperm obtained from the vas deferens and incubated in seminal plasma (PS), a ionic medium similar to seminal plasma (MA) and HTF-HEPES culture medium. MATERIAL AND METHOD: Aspirates from de proximal vas deferens were obtained in eight healthy men during vasectomy and separated in three aliquots (5-6 millions sperm/mL). Aliquots were incubated on PS, MA and HTF-HEPES. Quantitative and qualitative motility were assessed at one hour intervals. RESULTS: Sperm motility decreased 30% in the 1st hour but at the 3rd hour it increased 70% in aliquots incubated in PS and MA. Straight line velocity (VSL) also increased significatively at the 1st and 3rd hour only in aliquots incubated in PS and MA compared to aliquots incubated in HTF-HEPES (1st hour: p < 0.01; 3rd hour: p < 0.001). Curvilinear velocity (VCL) decreased at 3 hours in aliquots incubated in PS and MA compared to HTF-HEPES (P < 0.001). VCL was lower on aliquots incubated in PS compared to MA (p < 0.01). CONCLUSIONS: Sperm activation occurred in PS and MA at 3 hours. Incubation in HTF-HEPES culture medium resulted in a progressive decrease of sperm motility. This suggests that ionic/osmolar composition of PS is important for sperm activation of sperm obtained from the vas deferens.

Culture Media↗
PubMed1989

[Effects of different buffers, ethanol and hydrogen peroxide on the morphology of cultured brain cells].

Effects of different buffers: N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), Ethanolamine (2-Aminoethanol), sodium bicarbonate (NaHCO3) in the presence of 0.003% H2O2 or 0.5% ethanol were investigated on cell cultures from brain (chick embryo 5 1/2 or 9 day in ovo). Morphological parameters: density of cells, reaggregation, size of cell aggregates were used. H2O2 in the presence of NaHCO3 led to a decrease of isolated cells and an increase of reaggregation with degeneration in the great clusters. When H2O2 and 5 mM HEPES were present together in the absence of NaHCO3, the aggregation was less than with HEPES alone. By use of a chemical defined medium DME/F-12 supplemented with Insulin, Transferrin, sodium selenite, bovine serum albumin and 15 mM HEPES both the number and the diameter of cell aggregates increased. Addition of 0.5% ethanol in the presence of NaHCO3 stimulated in cultures of the ganglion trigeminale and telencephalon (9 day chick embryos) the regeneration of neurites and the proliferation and migration of non-neural cells. No growth of neurites was observed on the same condition in brain cultures from 6 day chick embryo. The number of large aggregates increased significantly in the presence of NaHCO3 and in the chemical defined and supplemented medium DME/F-12. Bologa et al. (1987) demonstrated that the buffers TREA (Triethanolamine) and HEPES enhance the expression of properties related to post-mitotic differentiated cellular states in both neurons and glia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

The effects of different buffers on the oxidation of DNA by thiols and ferric iron.

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe3+ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe3+ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe3+ and O2 resulted in the formation of an oxidant capable of producing 8-hydroxy-2'-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe3+ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Influence of pH on formation and stability of phosphatidylcholine/phosphatidylserine coatings in fused-silica capillaries.

The effect of pH on the formation and stability of phospholipid coatings in fused-silica capillaries in electrophoresis was investigated. A liposome solution consisting of 3 mM of 80:20 mol% phosphatidylcholine/phosphatidylserine (PC/PS) in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer was used as coating material. The coating was prepared by a method described earlier and five steroids were used as neutral model analytes. First, the effect of pH of the coating solution on the formation and stability of phospholipid coatings was studied at pH 6.5-8.5. The pH of the background electrolyte (BGE) solution (HEPES) was either kept constant at pH 7.4 or made similar to the pH of the liposome coating solution. Results showed that attachment of the coating on the fused-silica wall mostly depends on the protonation of amines of the phospholipids and HEPES. The ability of the phospholipid coating to withstand changes in pH was then investigated by coating at pH 7.5 and separating steroids with acetic acid, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, or glycine BGE, adjusted to pH between 4.5 and 10.8. The results showed that with use of BGE solution at pH 10.8, the separation of steroids was not successful and the electroosmotic flow was high because of leakage of the phospholipid coating during preconditioning of the capillary with BGE solution. There was no phospholipid leakage with a BGE solution of pH 4.5, indicating that the protonated form of the functional groups of PS and HEPES participating in the attachment of the phospholipid coating to the capillary play an essential role in the success of the coating.

Chromatography↗