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PubMed1982

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes.

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.

Alkanesulfonates↗
PubMed1991

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) does not modify the angiotensin II-stimulated calcium signal in cultured bovine glomerulosa cells.

Studies were performed to determine if the sustained elevation in [Ca2+]c noted previously in glomerulosa cells in response to Ang II resulted from the presence of HEPES in the experimental medium. At confluence, primary cultures of bovine glomerulosa cells were maintained for 24-30 h in the presence of either 14 mM NaHCO3/5% CO2 or 25 mM HEPES/4 mM NaHCO3/air. During subsequent experimental periods, cells were incubated in the presence of the corresponding or reciprocal buffer, and the effects of Ang II on [Ca2+]c were monitored by fura 2 fluorescence. Increases in [Ca2+]c produced by Ang II in cells continuously maintained in either HCO3(-) - or HEPES-buffered media were similar, and with the same monolayer the nature of the Ang II-stimulated Ca2+ signal was independent of the buffer employed. Moreover, the Ang II-stimulated Ca2+ signal was not significantly affected by the removal of HCO3- from the superfusate. These results indicate that the sustained increase in [Ca2+]c is not an artifact introduced by the use of HEPES as an experimental buffer, but rather a normal component of the Ang II-stimulated Ca2+ signal.

Aldosterone↗
PubMed1980

Adverse effects of Tris. HEPES and MOPS buffers on contractile responses of arterial and venous smooth muscle induced by prostaglandins.

The influence of artificial buffers, Tris, HEPES and MOPS, on PGA1, B2 and F2 alpha-induced contractile responses of isolated rat aorta and portal vein was investigated. All three buffers, in 5mM concentration, when substituted for bicarbonate and phosphate anions, differentially attenuated PG-induced contractile responses; the vein responses exhibited the greater sensitivity to artificial buffer inhibition. In the presence of the artificial buffers, the PG-induced fast phase contractile components on the arterial smooth muscle were obliterated and the slow, tonic components were markedly depressed. PG contractile concentration-effect curves were shifted, differentially, to the right, concomitant with differential reductions in maximum tensions. Reincubation of the vascular tissues in normal Krebs-Ringer bicarbonate for 30-60 min resulted in a complete restoration of full PG-induced contractions. These results support the idea that Tris, HEPES and MOPS may interfere with the binding, translocation and utilization of calcium ions in vascular smooth muscle cells. One must be cautious regarding conclusions drawn for calcium-dependent responses obtained in smooth muscles in which Tris, HEPES or MOPS was the sole buffering agent.

Alkanesulfonates↗
PubMed1992

Regulation of lipoprotein lipase by dibutyryl cAMP, cholera toxin, Hepes and heparin in F1 heart-cell cultures.

Regulation of lipoprotein lipase was studied in mesenchymal rat heart-cell cultures. Treatment of the cultures with dibutyryl cyclic AMP or with cholera toxin resulted in an increase in LPL activity and a comparable increase in LPL mRNA. When the cells were exposed to 100 mM Hepes for 24 h, total enzyme activity rose 2-fold and LPL mRNA increased 2.4-fold. After 72 h, there was a 3-fold increase in LPL mRNA and a 4-fold rise in cellular LPL activity, while medium activity increased 20-fold. Exposure of the cultures to heparin for 24 h resulted in a 3.2-fold increase in total activity and a 36-fold increase in medium activity. This increase was not accompanied by any rise in LPL mRNA. Addition of actinomycin D to control dishes for 24 h resulted in a 33% reduction in LPL mRNA and a 43% reduction in enzyme activity. These values were 71% and 56%, respectively, in Hepes-treated cells, indicating that no stabilization of LPL mRNA occurred under these conditions. It can be concluded that in mesenchymal rat heart-cells in culture cAMP and cholera toxin upregulate lipoprotein lipase at the level of transcription. The increase in LPL activity after 24 h exposure to Hepes could be compatible with transcriptional regulation, while exposure to heparin is not accompanied by a change in LPL mRNA.

Animals↗
PubMed2005

Effects of Tris and Hepes buffers on the interaction of palladium-diaminopropane complexes with DNA.

The Pd(II) complexes, [PdCl(2)(1,2-pn)] and [PdCl(2)(1,3-pn)] (pn is diaminopropane), were synthesized and characterized by analytical and spectroscopic (FT-IR, (1)H NMR and (13)C NMR) techniques. UV difference spectral study performed on Pd-pn/DNA systems, indicate a pronounced interaction of palladium complexes with DNA in cell-free media; comparison of lambda(max), Abs(max) and %H values observed for the two compounds might be attributed to structural differences of the chelated ligand rings. Results obtained from electrophoretic analysis of Pd complexes in presence of pBR322 plasmid DNA show a clear decreasing of the supercoiled (SC) DNA form mobility, that could be attributed to unwinding of the double helix; a parallel increasing of the open-circular (OC) DNA form mobility is also noted, this fact implying that the binding of complexes either shortens or condenses the DNA helix. Interaction studies of Pd complexes with plasmid DNA in different buffer systems indicate that DNA binding efficiency capable of modifying the tertiary structure of pBR322 decreased from NaClO(4) to Hepes 2, Hepes 1 [Hepes=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and Tris [(hydroxymethyl)aminomethane] buffers, in this order. Moreover, the level of DNA modifications produced by palladium complexes in 10 mM NaClO(4) remains unchanged after transferring the samples into the medium required for subsequent biophysical or biochemical analyses.

Animals↗
PubMed2001

The influence of HEPES on light responses of rabbit horizontal cells.

HEPES-buffered solutions, mostly used in studies of isolated cells, and bicarbonate-buffered solutions, mostly used in studies of isolated retinal tissues, have both been used to superfuse an isolated rabbit retina preparation. The responses of horizontal cells (HCs) to light, detected by intracellular microelectrodes filled with Lucifer Yellow, were recorded. Buffering of the superfusate with 100% HEPES completely, but reversibly, abolished the responses of A-type HCs, and is not, therefore, suitable for studies on isolated rabbit retinas. The responses remained when buffering was partially with HEPES and partially with bicarbonate, but were changed: in A-type HCs the overshoot was reduced and the afterpotential was increased. The overshoot may be caused by feedback of HCs on the cones and might be dependent on pHi at the synaptic structure between HCs and photoreceptors.

Animals↗
PubMedDate not supplied

Effects of bicarbonate versus HEPES buffering on measured properties of neurons in the salamander retina.

Electrophysiological studies of the isolated retina involve perfusing the tissue with a physiological Ringer's. Organic pH buffers such as HEPES have become increasingly popular in recent years because for many purposes they offer a convenient and reliable alternative to the more traditional bicarbonate/CO2. In this paper, however, we report that important functional properties of rods, bipolar cells, and horizontal cells in the salamander, Ambystoma tigrinum, are sensitive to the choice of buffer and, in the case of horizontal cells, that sensitivity is acute. In bicarbonate/CO2 Ringer's, the dark potential of the horizontal cell was typically near -50 mV and saturating light caused it to hyperpolarize to about -75 mV. On switching to HEPES-buffered Ringer's at the same pH, horizontal cells depolarized in darkness to about -20 mV, close to the chloride equilibrium potential, and the kinetics of their light responses changed. The cone-driven components of light responses increased in size relative to rod-driven components. Saturating lights still hyperpolarized the cells to -75 mV, however. Horizontal cells, being coupled via gap junctions, form a syncytium and syncytial length constants, measured in bicarbonate/CO2 Ringer's, were generally in the range 150-225 microm. On switching to HEPES-buffered Ringer's, length constants increased substantially to 250-330 microm. All these changes were reversible. We discuss our findings within the context of the cell's ability to regulate its internal pH.

Ambystoma↗
PubMed1988

Free-radical generation by copper ions and hydrogen peroxide. Stimulation by Hepes buffer.

Hydroxyl radicals (OH.), generated by a phosphate-buffered Cu2+/H2O2 system, were detected by lucigenin-amplified chemiluminescence, deoxyribose degradation and benzoate hydroxylation. In each system the buffer, Hepes, was found to stimulate radical generation significantly. There are two main reasons for this effect: Hepes increases Cu2+ solubility in phosphate-buffered systems, and forms a complex with Cu2+ that is effective in generating OH. from H2O2. Pipes, a structurally similar buffer, and histidine, a known Cu2+ chelator, were found to have a similar effect. These data suggest that the crucial factor in such free-radical-generating systems is the availability of Cu2+, and that these actions of Hepes should be considered in the design of studies utilizing such systems.

Benzoates↗
PubMed2001

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures.

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).

Animals↗
PubMed1992

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function.

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.

Binding Sites↗
PubMed1995

In vitro effect of Hepes buffer on maintaining the number of gap junction plaques in human myometrium at term.

In human myometrium, at various stages of labour and in correlation with the concentration of progesterone and estradiol in maternal blood the formation of gap junctions has been described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated human myometrium at term to Hepes buffer, on the number of gap junction plaques. Biopsies of myometrium were obtained from 5 pregnant women at term: they had an elective caesarean operation in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer, trimmed under a stereo microscope into small strips and kept in warm (37 degrees C) oxygenated Hepes buffer supplemented with glucose (0.01 mM). Then some strips of myometrium were incubated at 37 degrees C for 10, 20, 40, 55, and 180 min. The number of gap junction plaques were counted: it decreased in strips of myometrium after 10, 20, 40, 55, and 180 min of in vitro experiment (P < 0.01 vs control). In some of our experiments, the decrease in quantity of gap junction plaques was very dramatic and far below 50% of their numbers found in control specimens but never reached the zero value.

Biopsy↗
PubMed1994

In vitro evaluation of phosphate, bicarbonate, and Hepes buffered storage solutions on hypothermic injury to immature myocytes.

In this study we evaluated cardiac myocyte viability and function under hypothermic conditions using three types of buffer solutions: phosphate buffer solution (PBS), Krebs-Henseleit bicarbonate buffer solution (KHB), and Hepes buffered minimum salt solution (MSS). As a control, normal saline solution (NSS) was used. Cardiac myocytes were isolated from neonatal rat ventricles. Myocytes (12.5 x 10(5) myocytes/culture flask) were then incubated at 4 degrees C for 6, 12, 18, and 24 hours in various buffer solutions. After each incubation time, CPK and LDH were measured. The myocytes were then incubated for an additional 24 hours at 37 degrees C to evaluate the recovery of the myocyte beating rate. Group MSS had a significantly better beating rate recovery than group NSS (control) after 18 hours (MSS, 32.7%, NSS, 0.0% of control; i.e., beating rate prior to hypothermic incubation). In contrast, group KHB showed a significantly lower recovery ratio than group NSS at 12 hours (41.0%, 78.8%, respectively), and the lowest recovery was observed in group PBS beginning at 6 hours of hypothermic incubation (27.6%). Group MSS significantly suppressed the release of CPK and LDH compared to group NSS at 24 hours (MSS, 246.7 and 440.2 mIU/flask; NSS, 369.7 and 821.3 mIU/flask, respectively). In contrast, groups PBS and KHB showed significantly increased CPK and LDH levels compared to group NSS after 12 hours (PBS, 388.6 and 721.4 mIU/flask; KHB, 340.5 and 540.5 mIU/flask; NSS, 91.5 and 222.7 mIU/flask, respectively). In conclusion, Hepes buffer has cytoprotective characteristics that may be suitable for long-term hypothermic preservation of immature myocardium compared to phosphate or bicarbonate buffer.

Animals↗
PubMed1985

HEPES may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites.

Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth.

Animals↗
PubMed1988

Fictive locomotion in the lamprey; a comparison using bicarbonate and HEPES buffers.

Fictive locomotion in the lamprey, elicited by N-methyl-DL-aspartate or D-glutamate, was compared in two buffer solutions, one using bicarbonate, the other using HEPES. No differences were observed in burst proportion, cycle length, or phase lag per segment between these buffers solutions. These data indicate that HEPES-buffered artificial cerebrospinal fluid, which does not require gassing with carbon dioxide to maintain pH, may be a suitable alternative to bicarbonate-buffered artificial cerebrospinal fluid.

Animals↗
PubMed1990

Effect of Tris, HEPES, and TES buffers on binding at mu-, delta-, and kappa-opioid sites in guinea pig brain.

In homogenates of guinea-pig brain minus cerebellum, the delta-binding of 1.5 nM [3H]-[D-Pen2,D-Pen5]enkephalin is little affected by the type and concentration of the three buffers, Tris-HCl, HEPES-KOH, or TES-KOH (10-75 mM). However, the mu-binding of 1 nM [3H]-[D]Ala2,MePhe4,Gly-ol5]enkephalin or the kappa-binding of 1.5 nM [3H]-U-69,593 is influenced by the choice of buffer. A suitable concentration of buffer for further analyses of opioid binding has been found to be 10 mM. At each site, the effects of MgCl2 on binding are the same whether 10 mM Tris-HCl, HEPES-KOH, or TES-KOH are used but variations of the effects of NaCl confirm the view that mu- and kappa-sites, but not delta-sites, are affected by choice of buffer. Furthermore, under some assay conditions the effects of NaCl and MgCl2 at the kappa-sites of guinea-pig cerebellum differ from their effects in brain minus cerebellum, indicating that these are differences of binding characteristics at the kappa-sites of these tissues.

Animals↗
PubMed2002

Voltage-dependent transient currents of human and rat 5-HT transporters (SERT) are blocked by HEPES and ion channel ligands.

The hyperpolarization-activated transient current of mammalian 5-hydroxytryptamine transporters (SERT) expressed in Xenopus oocytes was studied. Human (h) and rat (r) SERT transient currents are blocked by HEPES with changes in the waveform kinetics, and the blockade of hSERT has use-dependent properties. HEPES also changes the time course of the prepriming step, especially for hSERT. Transient currents at hSERT and rSERT are also blocked by spermine and spermidine in the mM range, and by fluoxetine, cocaine, QX-314, and QX-222 in the microM range. These pharmacological and kinetic properties of transient current blockade emphasize the similarities between the transient current and phenomena at ion channels.

Animals↗
PubMed1998

Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors.

We investigated the effects of organic buffers on the NO-like biological activities of ONOO-. In HEPES buffer (50 mM), ONOO- (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO. ONOO- produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM). Electrochemical detection of NO showed that the biological effects of ONOO- in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions. CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway. Formation of NO from ONOO- was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO- on cGMP accumulation in this buffer. These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO-.

Animals↗
PubMedDate not supplied

Effects of TRIS and HEPES on function of rabbit muscle light sarcoplasmic reticulum.

ATP hydrolysis activity and calcium transport activity were determined on light sarcoplasmic reticulum from rabbit skeletal muscle. The effects of two buffers, TRIS and HEPES, were compared. Titration of TRIS into sarcoplasmic reticulum preparations in HEPES provided evidence for TRIS inhibition of ATPase activity and TRIS stimulation of calcium transport activity.

Adenosine Triphosphate↗