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PubMed1997

[Optimum choice for the cultivation of CFU-GM in media buffered with HEPES].

The effects of media buffered with HEPES of different concentrations and different incubation periods on the proliferation and differentiation of CFU-GM were studied under the standard condition for culture. The results exhibited that when the incubation period prolonged, the pH values of culture system raised and the colony formation of CFU-GM decreased; the culture system added with proper HEPES buffer solution could maintain and increase the colony formation of CFU-GM, especially the culture system in which the pH value was not adjusted before the experiment. We also found that media buffered with HEPES could change the ratio of different colony types. This showed that media buffered with HEPES could not only stimulate the proliferation of CFU-GM, but also affect its differentiation. The results suggest that media buffered with HEPES are better than those without HEPES for the culture of hematopoietic progenitor cells, and the optimum concentration is 10-20 mmol.L-1.

Animals↗
PubMed1995

Influence of Hepes- and CO2/HCO(3-)-buffer on Ca2+ transients induced by TRH and elevated K+ in rat pituitary GH4C1 cells.

The influence of two buffer systems (Hepes and CO2/HCO3-) on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH and by elevated K+ were studied in single, and small clusters of, clonal rat pituitary GH4C1 cells using Fura 2. The steady-state level of [Ca2+]i was virtually identical in Hepes and CO2/HCO3-. In both buffers, addition of TRH induced a transient increase in [Ca2+]i which attained a significantly higher peak in Hepes (357 +/- 43 nM) when compared with values measured in the presence of CO2/HCO3- (184 +/- 21 nM). In Hepes, the basal IP3-level was higher than in CO2/HCO3-. The TRH-evoked increase in IP3 was higher in magnitude in Hepes than in CO2/HCO3-, although the stimulated/basal ratio was not different between the two buffers. The buffer composition had no effect on the specific binding of 3H-TRH to the cells. Furthermore, the amplitude of the increase in [Ca2+]i evoked by 50 mM K+ was identical in both buffers. TRH and K+ had no effect on pHi in either buffer. The present results indicate that HCO3- has an influence on TRH-induced Ca2+ transient, at least in part by modifying the TRH-evoked production of IP3.

Animals↗
PubMed1986

Growth of mouse embryos in bicarbonate media buffered by carbon dioxide, hepes, or phosphate.

The purpose of this study was to determine if mouse embryos could be grown successfully in a culture medium devoid of the carbon dioxide phase (CO2). Mouse embryos fertilized in vivo were collected and cultured in Hepes medium with and without bicarbonate (HCO3-) and a phosphate medium with and without HCO3-. In these experiments no CO2 gas phase was used. Further embryos were cultured in Whittingham's modified Tyrode's (T6) medium with a CO2 gas phase and served as controls. The degree of embryonic development was noted. Surviving blastocysts were transferred to the uteri of pseudopregnant mice and delivery at term was allowed to occur. There was no significant difference in the degree of embryonic development in those embryos cultured in T6 or Hepes medium (+ HCO3-) or in the number of live offspring obtained when these blastocysts were placed within the mouse uterus. Although embryonic development apparently proceeded successfully in the phosphate (+ HCO3-) medium, none of these blastocysts survived when transferred to mouse uteri. No embryonic growth occurred in either the Hepes or phosphate media which were devoid of HCO3-. It appears that a Hepes medium containing HCO3-, which uses no CO2 gas phase, is as effective as T6 medium, which uses a gas phase, in supporting in vitro mouse embryonic growth.

Animals↗
PubMedDate not supplied

Incorporation of labeled glucosamine into glycoproteins by organ cultures of hamster trachea: adverse effects of HEPES buffer.

Hamster tracheas were cultured in serum-free CMRL 1066 medium buffered with either NaHCO3 alone or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) plus NaHCO3 in an atmosphere of 95% O2 and 5% CO2. Afther 2 days in culture, tracheas maintained in HEPES plus NaHCO3-buffered medium showed an altered surface morphology. Histiological examination after 6 days of culture with HEPES plus NaHCO3 showed the presence of only scattered clumps of cilia. Incorporation of [14C]-glucosamine into intracellular glycoproteins was reduced by 75% and into secreted glycoproteins by 54% in cultures buffered with HEPES plus NaHCO3, compared to NaHCO3-buffered cultures. Incorporation of [H]fucose into intracellular glycoproteins was also reduced, although no effect was observed on secreted glycoproteins.

Animals↗
PubMed1985

Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium.

The addition of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) to RPMI 1640 medium markedly increases the production of cytotoxic products during exposure of the medium to visible light. The cytotoxicity has been analyzed by measuring uptake of [3H]thymidine by murine thymocytes cultured in preirradiated medium containing 25 mM HEPES. Complete inhibition of thymidine uptake was produced by exposing 50% of the culture medium to light for 3 h before addition of cells. The HEPES-mediated effect requires only that HEPES and riboflavin be exposed to light; other medium constituents are not necessary. Hydrogen peroxide is a principal cytotoxic agent produced in this system. It is demonstrated that most, but not all, of the inhibition of thymidine uptake can be attributed to hydrogen peroxide.

Animals↗
PubMed1983

Effect of Hepes on the fibroblast cerebroside sulfate loading test.

The intact fibroblast cerebroside sulfate loading test is useful because sulfatide hydrolysis can be demonstrated in late onset MLD cell types with 1% or less of normal arylsulfatase A. In such cells, hydrolysis of sulfatide was inhibited when the loading test was carried out in growth media containing the organic ampholyte Hepes. Since Hepes did not affect uptake of sulfatide nor intracellular levels of arylsulfatase A, it was concluded that Hepes inhibited sulfatide hydrolysis by increasing lysosomal pH. The cerebroside sulfate loading test in the presence of Hepes should be useful as a probe for arylsulfatase A dysfunction in atypical MLD fibroblasts.

Animals↗
PubMed1996

Ionic basis of the membrane potential responses of rat dorsal vagal motoneurones to HEPES buffer.

The effects of 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffered artificial cerebrospinal fluid (aCSF) on membrane potential and the action potential were studied in 93 dorsal vagal motoneurones (DVMs) using an in vitro slice preparation of the rat medulla. Changing from bicarbonate/CO2 aCSF to HEPES aCSF resulted in a depolarisation of 6.0 +/- 0.6 mV and an increase in input resistance (RIn; n = 61). In the presence of 5 mM 4-AP, HEPES either had little effect (n = 9) or hyperpolarised the membrane (n = 10). Mn2+ (3 mM) or Ni2+ (200 microM) abolished the hyperpolarisation and its associated increase in RIn. In voltage-clamp studies 5 mM 4-AP eliminated a transient outward current and Ni2+ blocked an inactivating inward current. It is concluded that HEPES buffer reduces the contribution of the A current to resting membrane potential and also reduces a Ni(2+)-sensitive transient ICa.

4-Aminopyridine↗
PubMed1990

Effect of HEPES on the Na+,Cl-dependent uptake of taurine and beta-alanine by cultured glial cells. Modulation by composition and osmolarity of medium.

The effect of HEPES on the Na+,Cl-dependent uptake of taurine and beta-alanine by cultured glial cells was investigated. Whatever the culture medium used, a similar inhibition of the uptake of taurine by HEPES was found. In a previous paper it was shown that this buffer was able to inhibit the uptake of taurine through a slow and a fast "mechanism", when present in the culture or in the incubation medium, respectively. Comparison of the effect of HEPES on the uptake of taurine and beta-alanine suggested that only the slow "mechanism" was common to the uptake of the beta-aminoacid. Therefore, the uptake of the beta-aminoacid might be affected by HEPES. On the other hand, a small increase of the osmolarity of the media altered the uptake of taurine, suggesting that caution is needed when interpreting pharmacological experiments.

Alanine↗
PubMed2005

Cu(II) complexation by "non-coordinating" N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer).

The combined potentiometric and spectroscopic studies of interactions of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with Cu(II) demonstrated that this popular buffer, commonly labelled as "non-coordinating" forms a CuL+ complex, with the logbeta(CuL) value of 3.22. This complex undergoes alkaline hydrolysis above pH 6, resulting in Cu(OH)2 precipitation. However, the presence of HEPES at a typical concentration of 100 mM at pH 7.4 elevates the apparent binding constant, being determined for a complex of another ligand, by a factor of 80. HEPES does not form ternary complexes with aminoacids Ala, Trp, and His, but may do so with other bioligands, such as nucleotides. Therefore, HEPES can still be recommended for Cu(II) studies in place of other common buffers, such as Tris and phosphate, but appropriate corrections and precautions should be applied in quantitative experiments.

Amino Acids↗
PubMed1996

Inhibitory effect of internal sodium and Hepes on the voltage-dependent potassium conductance of rat Leydig cells.

We used the whole-cell configuration of the patch-clamp technique to investigate modulation of the voltage-dependent K+ conductance of rat Leydig cells by intracellular pH, Hepes, and Na+. pH (range 6.8-7.4) has no effect on current. Removal of Na+ (from 10 to 0 mM) increases the amplitude of K+ current and abolishes the plateau effect on peak current. Reduction of Hepes (from 25 to 10 mM) increases the amplitude of K+ current and, simultaneously, shifts the threshold for current activation to the left, but the plateau of current is still present. Removal of Na+ cancels the potentiating effect of Hepes on frequency of occurrence of time-dependent inactivation of K+ current. We conclude that the blocking effect of Hepes and Na+ on K+ current may presumably involve two distinct mechanisms, but we can not exclude some cooperativity in their ability to inactivate the voltage-gated potassium conductance of rat Leydig cells.

Animals↗
PubMed2000

Beneficial effect of HEPES buffer in repeated coronary reperfusion.

BACKGROUND: We have demonstrated previously that, when repeated reperfusion is performed after reocclusion, there is a decrease in the amount of myocardial salvage, despite early reperfusion. Treatment with nisoldipine induced a beneficial effect by reduction of infarct size in this experimental model. OBJECTIVE: To study the effect of HEPES buffer on infarct size, using a repeated-reperfusion model. METHODS: The left anterior descending coronary artery was occluded in anesthetized dogs. Thirty minutes after the occlusion, dogs were allocated randomly to either the treatment group (n = 7; HEPES 0.64 mmol/l infused intravenously throughout the experiment) or the control group (n = 8; saline). Occlusion was maintained for 2 h, followed by 1 h of reperfusion, then 1 h of reocclusion and 2 h of second reperfusion. An in-vivo area at risk was determined by gentian violet staining, and infarct size was defined and quantitated by triphenyl-tetrazolium chloride staining. RESULTS: Hemodynamic measurements were similar in both groups. Mass of necrosis/mass at risk was significantly smaller in the HEPES group (30.7 +/- 1.6%, mean +/- SEM) compared with controls (50.6 +/- 3.8%, P < 0.001). CONCLUSION: Treatment with HEPES induces a beneficial effect by reduction of infarct size in repeated coronary reperfusion.

Analysis of Variance↗
PubMed1983

Comparison of portal vein responsiveness in Tris, HEPES or bicarbonate-phosphate buffered media.

The effects of using Tris or HEPES buffered solutions instead of bicarbonate-phosphate buffered solution on the spontaneous activity of the rat portal vein were studied. The effect of the solution buffered with Tris on the spontaneous contractile activity was a minor change in amplitude and frequency, increasing the integrated isometric force by about 20%. A small increase in the mechanical response to variations in [Ca2+]0 (1.0-2.5 mM) was also observed. The response to field stimulation of intramural nerves was significantly lowered in Tris but the response to exogenous noradrenaline was not affected. HEPES buffer does not significantly affect the spontaneous activity, the concentration-effect curve for Ca2+, the response to field stimulation or to exogenous noradrenaline. Transient changes in the spontaneous activity and the resting membrane potential were observed when the solution was changed from Krebs to Tris (or HEPES) solution or vice versa. These changes disappear if the Tris or HEPES solutions were aerated with 4% CO2 in O2 (at pH 7.4) instead of pure O2. A small change in extracellular pH (+/- 0.2 pH units) significantly affects the spontaneous activity. A change in pH may be the reason for the observed effects in the different buffer media.

Animals↗
PubMed1984

Effect of HEPES buffer on corneal storage in MK medium.

Rabbit corneas were stored for 7 days in either MK medium containing gentamicin or modified MK medium containing HEPES buffer, gentamicin and phenol red. Corneas stored for 7 days in modified MK medium were thicker than corneas stored in MK medium. Corneal endothelial permeability to inulin and dextran was similar following 7 days of storage in either solution. Transmission electron microscopy of corneal endothelial cells stored in either solution showed intact cell membranes and organelles. In vitro perfusion of rabbit corneas in the specular microscope with Krebs Ringer bicarbonate containing HEPES buffer swelled at 17 +/- 1 micron/h, whereas those perfused with Krebs Ringer bicarbonate alone swelled at 7 +/- micron/h. Perfusion with Krebs Ringer bicarbonate containing phenol red did not result in an increased corneal swelling rate. The work indicates that HEPES buffer has an adverse effect on corneal endothelial pumping function, and this results in corneal swelling during storage as well as during perfusion in the specular microscope. The adverse effect appears to be, at least in part, transient: however, the ultimate, long term effect of HEPES buffer on corneas stored prior to penetrating keratoplasty is not known and deserves continued investigation.

Animals↗
PubMed1987

Comparison of Ham's F10 with CO2 or Hepes buffer for storage of equine embryos at 5 C for 24 H.

Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM). Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron). Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare. Twenty embryos cultured in Dulbecco's phosphate buffered saline + 10% FCS and transferred less than 1 h after collection were used as controls. Pregnancy rates were higher (P less than .05) for embryos stored in Ham's F10 + CO2 (70%, 55%) than for embryos stored in Ham's F10 + Hepes (20%, 15%) at 14 and 35 d, respectively. At 14 d, pregnancy rates for control embryos (90%) were similar (P greater than .05) to pregnancy rates for embryos cultured in Ham's F10 + CO2 (70%); however, by 35 d, pregnancy rates were higher (P less than .05) for controls (80%) than for embryos stored in Ham's F10 + CO2 (55%). It was concluded that Ham's F10 + CO2 was superior to Ham's F10 + Hepes for short-term storage of equine embryos at 5 C, and that satisfactory pregnancy rates could be obtained from transfer of embryos stored in Ham's F10 + CO2 at 5 C for 24 h.

Animals↗
PubMed2002

Acidic HEPES-KH reperfusion enhances myocardial protection in immature rabbits.

To study the effects of different pH HEPES-KH reperfusate solution on immature myocardial protection, isolated perfused Langendorff model from immature rabbit hearts were developed formed. Control group (C) was perfused only with pH 7.4 HEPES-KH solution for 90 min. Ischemia/reperfusion group (group I/R) was perfused with pH 7.4 HEPES-KH solution before ischemia or after ischemia. Experimental group (group E), after ischemia, was perfused with pH 6.8, pH 7.1 and pH 7.4 HEPES-KH solutions for 5 min, 5 min, and 20 min, respectively. The left ventricular function recovery, MWC, LDH and CK leakage, MDA, ATP content, and SOD activity were determined. Our results showed that the left ventricular function recovery, ATP content and SOD activity in group E were higher than those of group I/R (P < 0.05). MWC, MDA content, LDH and CK leakage in group E were lower than those of group I/R (P < 0.05). These findings suggested that pH paradox might be one of important mechanisms for immature myocardial ischemia-reperfusion injury, and acidic perfusate, at the beginning of reperfusion, might attenuate pH paradox and ameliorate functional recovery in isolated perfused immature rabbit hearts.

Animals↗
PubMed1989

Effect of HEPES on the taurine uptake by cultured glial cells.

HEPES inhibited the taurine uptake in glial cells. A different kind of inhibition was observed when HEPES was present in the culture medium or in the incubation medium used for the taurine-uptake measurement. As an example of a possible interference of HEPES in pharmacological experiments, we have studied the effect of this buffer on the modulation of taurine uptake by beta agonists or ionic concentration.

Animals↗
PubMed1990

In vitro fertilization (IVF) of mouse ova in HEPES-buffered culture media.

Some major drawbacks of a bicarbonate-buffered culture medium include the requirement of an elaborate incubator system able to maintain a 5% CO2 environment and the inability of the culture medium to maintain a physiological pH range (pH 7.3-7.4) in room air (0.03% CO2). This work resulted in the development of IVF culture media, BB (modified T6) and Hams-HEPES, which use HEPES-buffered systems not requiring the specialized CO2 environment to maintain a physiological pH range in room air. These media generate above-average cleavage rates in in vitro fertilized, superovulated B6CBAF1 mice ova. The effect of heparin and HEPES on cleavage was studied and neither had a significant effect at the concentrations used. Cleavage rates of nonfertilized ova (parthenogenic division) were 9 to 13%. There was no significant difference in parthenogenesis between any of the culture media and it appears to be a function of the strain of mice and the timing between human chorionic gonadotropin (hCG) injection and ovum collection. These results emphasize the need to account for parthenogenesis when determining cleavage rates of in vitro fertilized mouse ova. Also, the results suggest that because of individual mouse differences in cleavage rates, it is important to use an adequate number of mice per group to determine an accurate, average cleavage rate.

Animals↗
PubMed1994

Vanadium induced hemolysis of vitamin E deficient erythrocytes in Hepes buffer.

Several vanadium compounds were tested for their ability to induce in vitro hemolysis of vitamin E-deficient hamster erythrocytes. Free vanadyl caused hemolysis in Hepes buffer but not in Tris or phosphate buffer, while hemolysis was inhibited by catalase, chelators such as deferoxamine mesylate and EDTA, and hydroxyl radical scavengers such as ethanol and D-mannitol. Although metavanadate itself could not induce hemolysis, metavanadate with NAD(P)H caused hemolysis in Hepes buffer only, and superoxide dismutase prevented it. Hydrogen peroxide, hydroxyl radical and Hepes radical were involved in vanadyl-induced hemolysis, superoxide anion was further involved in metavanadate plus NAD(P)H-induced hemolysis. Vitamin E prevented hemolysis under both conditions.

Analysis of Variance↗