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.     

Use of amine buffers for in vitro studies of bone resorption

Summary Responses of cultured fetal rat bones to parathyroid hormone (PTH) were compared in media gassed with 5% CO₂ and buffered with bicarbonate or in media buffered with the amine buffers HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)), Tris (tris(hydroxymethyl)aminomethane), or glycylglycine. PTH-induced bone resorption was markedly reduced in media buffered with the amine buffers and cultured in air in the absence of added bicarbonate. When the amine buffers were added to bicarbonate-buffered media, they decreased the responses of the bones to submaximal concentrations of PTH. But the response to supramaximal concentrations of PTH was usually not impaired. The results confirm our earlier studies indicating a CO₂ and/or bicarbonate requirement for optimal bone resorption. They also demonstrate that the amine buffers can inhibit the responses of bone to PTH even in the presence of CO₂ and bicarbonate. Supported by NIH research Grant #AM 11262. Supported by NIH Research Career Development Award #AM 70210.     

The effects of the buffer hepes on the division potential of WI-38 cells

Summary N-2-Hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) and N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) have been found to be nontoxic substitutes for bicarbonate buffer in cell culture media. WI-38 embryonic human lung cells have been carried beyond the 60th passage with both HEPES and TES buffers.     

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

Effects of media buffer systems on growth and electrophysiologic characteristics of cultured sweat duct cells

Summary Primary cultures of human reabsorptive sweat duct cells were grown in MCDB 170 medium buffered with either HEPES, bicarbonate, or a mixture of HEPES and bicarbonate buffers. Cultures grown in MCDB media containing bicarbonate seemed to differentiate into a multilayered, keratinized epithelium and began senescing after 1 wk in culture. In contrast, cultures grown in media containing HEPES as the only buffer seemed to undergo a selection process, resulting in the outgrowth of cells that did not multilayer or keratinize extensively for up to 3 or 4 wk in culture. Despite marked differences in growth, cells grown in both bicarbonate and HEPES-buffered media retained electrophysiologic characteristics appropriate to the progenitor. Mean resting potentials were −21.8±0.8 mV (n=82), −23.3±1.3 mV (n=70) and −18.2±0.8 mV (n=82) for duct cells grown in HEPES, bicarbonate, and HEPES-bicarbonate media, respectively. Substitution of Cl⁻ with the impermeant anion gluconate in the bathing medium caused membrane potential depolarization in all media, revealing the presence of a Cl⁻ conductance. Administration of the Na⁺ conductance inhibitor amiloride hyperpolarized the mean resting potential of cells grown in HEPES medium (−6.8±0.6 mV,n=68), bicarbonate medium (−6.9±0.5 mV,n=60), and HEPES-bicarbonate medium (−5.9±0.6 mV,n=69), demonstrating expression of a Na⁺ conductance. We observed some but minimal variation with age in any of these conditions. This work was supported by grant DK41329-02 from the National Institute of Health, Bethesda, MD, and a Postdoctoral Fellowship to Dr. Bell from the National Cystic Fibrosis Foundation.     

Stable adducts of nerve agents sarin,soman and cyclosarin with TRIS,TES and related buffer compounds—Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry

Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of these adducts as phosphonic diesters using 1D ¹H-³¹P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates with TRIS and TES are fast enough to compete with spontaneous hydrolysis in aqueous solution and to yield substantial amounts (up to 20–40%) of buffer adduct over the course of several hours. A reaction mechanism is proposed in which the amino function of the buffer serves as an intramolecular proton acceptor rendering the buffer hydroxyl groups nucleophilic enough for attack on the phosphorus atom of the agents. Results show that similar buffer adducts are formed with a range of hydroxyl and amino function containing buffers including TES, BES, TRIS, BIS-TRIS, BIS-TRIS propane, Tricine, Bicine, HEPES and triethanol amine. It is recommended to use alternative buffers like MOPS, MES and CHES when working with G-type nerve agents especially at higher concentrations and over prolonged times.     

Cartilaginous metaplasia of the thoracic aorta of control turkeys and exacerbation by beta-aminopropionitrile

Seventeen of sixty distal extremities of the thoracic aortas of 12-week-old control male turkeys and 37 of 40 distal extremities of the aortas of turkeys fed 0.07% beta-aminopropionitrile (BAPN) from 4 to 12 weeks of age contained areas of cartilaginous metaplasia when examined by light microscopy. The cartilaginous areas were generally elongated and located in the subendothelium of control turkeys, but a roundish area of cartilage was occasionally evident in the deep media. The magnitude of chondroplasia was enhanced by feeding BAPN; the extensive lesion usually extended from the subendothelium to deep in the media. Regardless of treatment, chondrocytes were pleomorphic, contained vacuoles, and had cytoplasmic processes. The cells were separated by pools of proteoglycans and connective tissue. The ultrastructure of chondrocytes in the aortas of both treatment groups was typical of this cell type. They had undulations or projections of the cell membranes. The cisternae of endoplasmic reticulum were dilated and contained electron-translucent material which was similar to extracellular proteoglycans. Golgi apparatus, free ribosomes, mitochondria, glycogen granules, filaments, and a centriole also were present in the cytoplasm. The extracellular matrix, which included collagenous and elastic fibers and also delicate fibrils and interconnecting matrix granules, separated adjacent chondrocytes by spaces of varying size.     

Immunohistochemical,autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein

Summary Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using ³⁵S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for ³⁵S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for ³⁵S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose

Primary cultures of embryonic chick sternal chondrocytes were embedded in a three-dimensional matrix of 1% solid agarose which was overlaid with nutrient media. The chondrocytes divided and formed nests of spherically shaped cells which were surrounded by an extensive extracellular matrix containing high molecular weight proteoglycans. Using light and electron microscopy, condensation of proteoglycan was observed pericellularly, often forming septa between cells of a nest, and as part of the outer boundary of the cell nest. No cross-striated collagen fibers were observed in the extracellular matrix although proteoglycan appeared to decorate a network of fine strands. Upon the addition of ascorbate to the nutrient media high molecular weight proteoglycans were synthesized, but there was a marked decrease in the synthesis of proteoglycans after a 10 day exposure to ascorbate. Morphologically, the decrease in proteoglycan synthesis was manifested in the discontinuous arrangement of the pericellular matrix as well as the diffuse form of the cell-nest boundary. Both of these structures were clearly defined in control cultures and were enriched in proteoglycan as demonstrated by ruthenium red staining. This study demonstrates that embryonic chondrocytes remain differentiated when cultured in solid agarose for a period of up to 15 days. They continue to synthesize their tissue specific macromolecules and are phenotypically stable when exposed to ascorbate for extended periods of time.     

Synthetic organic pH buffers can support fertilization of guinea pig eggs,but not as efficiently as bicarbonate buffer

Bicarbonate ions (HCO₃^?) in the medium are not absolutely essential for the fertilization of guinea pig eggs. However, fertilization takes place most efficiently in HCO₃^?-buffered medium. Capacitation, the acrosome reaction, and zona penetration by spermatozoa can occur in HCO₃^?-free media with synthetic organic buffers (i.e., MOPS, TES, HEPES, Tris, and TAPSO) but not as efficiently as in the HCO₃^?-buffered medium. It appears that HCO₃^? functions as more than just a pH-buffering molecule.     

ON THE SITE OF SULFATION IN THE CHONDROCYTE

As observed autoradiographically in the cartilage of embryonic rats, radiosulfate is bound and concentrated only in vesicles of the juxtanuclear Golgi apparatus of secreting chondrocytes within 3 minutes of its presentation. From this area, vacuoles migrate peripherally and lodge in the subcortex; their sulfated contents are thence discharged via stomata to the extracellular matrix. The label, apparently often associated with microvesicles at 10 and 20 minutes, is subsequently localized in the dense contents of the larger vacuoles. Bound radiosulfate is not detectable in other organelles. It is concluded that the vesicular component of the Golgi apparatus is the actual site of sulfation. Intracellular hyaluronidase-sensitive metachromatic granules are found chiefly at the cell periphery or mantle, rarely juxtanuclear in the main Golgi zone.     

Immunohistochemical, autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein.

Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using 35S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for 35S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for 35S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

An electron microscopic study of hemoglobin synthesis in the marine annelid,Amphitrite ornata (Polychaeta: Terebellidae)

The heart-body of the marine worm Amphitrite, located within the supraesophageal dorsal vessel, is in the form of a cylinder the thin wall of which is deeply corrugated by luminal projections and folds along its entire length. It is anchored in places to the luminal surface of the dorsal vessel by an extracellular matrix containing collagen fibers. The luminal surfaces of both the heart-body and the dorsal vessel are covered by a basement membrane-like vascular lamina which in turn supports a discontinuous pseudoendothelium of littoral hemocytes. The cells of the heart-body constitute a pseudostratified, high columnar epithelium. They possess extensive rough endoplasmic reticulum (RER), a well developed Golgi zone, ferritin particles and granules, and several types of membrane-bound inclusions. Hemoglobin molecules identical to those in the circulation lie within cytoplasmic, membrane-bound vesicles. Analysis of our electron micrographs suggests the following sequence of hemoglobin production and secretion: Large quantities of a moderately dense flocculent material, probably globin, are synthesized in RER and move to the Golgi zone within partly rough- and partly smooth-surfaced transitional cisternae; small transport vesicles, formed from Golgi cisternae that have fused with transitional cisternae, convey the flocculent material from the convex to the concave face of the Golgi complex; a similar flocculent material and an amorphous, highly dense material are processed in the Golgi complex and are transferred to condensing vacuoles in which clearly identifiable hemoglobin molecules are first observed. Mature secretory vesicles containing only hemoglobin migrate to the cell periphery and discharge their contents by exocytosis. Hemoglobin molecules then cross the vascular lamina to reach the circulation.     

Effect of ethanesulfonic acid buffers and pH on the accumulation of a nervous system-specific protein (S-100) and a glial-enriched enzyme in a clonal line of rat astrocytes (C6).

Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.     

A comparison of the mechanical performance of papillary muscle and trabeculae carneae of cat ventricle

The mechanical performance of papillary muscle and of trabeculae carneae from cat ventricle were compared in physiologic salt solutions using two different buffers, bicarbonate and HEPES. The results show that there are no significant differences between the muscle types and that, under the conditions of these studies, HEPES may be used interchangeably with bicarbonate.     

普通大气条件下的蚀斑试验

草鱼出血病病毒湖南邵阳株(CCHV—873),常规培养条件下能在鱼肾(CIK)细胞上形成直径约2mm的蚀斑。当采用三种缓冲系统(MFM-NaHCO_3、MEM-Tris、MEMHEPES)的培养液在普通大气条件下分别培养CIK细胞时,三天内培养液的pH略有变化,其变化范围在0.2—0.4左右,但细胞生长仍然良好,三者无明显差别。在上述系统,以双相法(培养液-凝胶)进行蚀斑试验时,观察到无机缓冲系统培养液的pH变化较大,有机缓冲系统则较稳定,且蚀斑形成的数量显著不同,后者效价比前者要高出4个数量级。因此,在培养液中加入适量的有机缓冲液代之以CO_2的调节是完全有可能的。     

HYDROGEN ION BUFFERING OF CULTURE MEDIA FOR ALGAE FROM MODERATELY ACIDIC,OLIGOTROPHIC WATERS 1

Five hydrogen ion buffers were compared for their usefulness in regulating pH in a model oligotrophic, moderately acidic (pH 6.0) algal growth medium. These were 3,3-dimethylglutaric acid (DMGA), tricarbaliylic acid (TCA), trans-aconitic acid (tAA), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 2-(N-morpholino) ethanesulfonic acid (MES). All buffers (2.5 mM) except HEPES limited the reduction of pH in a NH₄⁺-based medium during growth of Chrysochromulina breviturrita Nich. to less than 0.12 units, compared with more than 2 units in an unbuffered medium. Long term growth of C. breviturrita in these media was significantly inhibited (P < 0.05) by TCA and tAA. MES was able to control pH with the minimum amount of NaOH (1.0 mM) added to the medium to adjust to pH 6.0. Four of five bacterial isolates were capable of utilizing tAA as a sole organic-C source, and no isolate could metabolize HEPES or MES. No significant differences (P > 0.05) were found in the maximum growth rates of six algal species (from five classes) in a medium with or without MES buffer, although significantly greater cell yields of Ochromonas danica Prings. were obtained in the buffered medium. MES (pK₄=6.15) was considered to be the most useful buffer in the pH range 5.0–6.5, due to its biological inertness, buffering capacity, the minimal requirement for excess base to adjust pH and its minimal metal complexing ability.     

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.     

Inhibition of photosystem II activity by Cu++ ion. Choice of buffer and reagent is critical

Cupric ion (Cu⁺⁺) inhibits the rate of photosystem II electron transport and the intensity of the variable part of chl a fluorescence in isolated chloroplast thylakoids. The inhibition is markedly dependent on the nature of the buffer used in the assay medium. In MES and HEPES buffers, complete inhibition of photosystem II occurs at 30 M of Cu⁺⁺, while in Tricine no inhibition occurred even at 200 M Cu⁺⁺. In other buffers used (TES, Phosphate, Tris), the efficacy of Cu⁺⁺ inhibition is intermediate. The calculated binding constants are found to correspond to the observed I₅₀ values for the six buffers used. It is concluded that the previous reports on copper inhibition, where buffers have been used indiscriminately should be reconsidered. Certain reagents such as hydroxylamine, ascorbate and diphenyl carbazide, which react with Cu⁺⁺, should be avoided.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - DAD diaminodurene - DPC diphenyl carbazide - Fv variable chl fluorescence - HEPES N-2-hydroxyethyl piperazine sulfonic acid - I ₃₀ inhibitor concentration causing 30% inhibition of Fv - MES 2-(N-morpholino) ethane sulfonic acid - MV Methyl viologen - PS II Photosystem II - PS I Photosystem I - TES N-tris(hydroxymethyl)-methyl-2-amino sulfonic acid - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-tris(hydroxymethyl) ethylglycine - Tris N-tris(hydroxymethyl)amino ethane     

Colorimetric pH measurement of animal cell culture media

Most animal cell culture media can be buffered using bicarbonate and high pressure CO₂ in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO₂ pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.