HEPES-buffered Solution Research Articles

Design and synthesis of 1,4-benzothiazine hydrazide as selective and sensitive colorimetric and turn-on fluorometric sensor for Hg2+ detection in aqueous medium

A highly colorimetric and fluorimetric chemosensor, 3-oxo-[1,4]-benzothiazin-2-ylidene acetohydrazide (L2) is reported for the mercury detection. In HEPES-buffered solution (CH3CN: H2O, 1:2, v/v, pH 7.2), L2 showed a characteristic absorption peak at 340 nm, after addition of Hg2+ induces color change from light yellow to purple with significantly enhancement in absorbance at 340 nm and a new band centered at 550 nm with red-shift of 110 nm. Furthermore, the L2 exhibited high sensitivity and selectivity overall emission change of more than 100-fold fluorescence intensity enhancement towards Hg2+ ion with a 1:1 binding stoichiometry (1.938 × 103 M−1 binding constant within detection limit as low as 5.4 × 10−8 M) in water samples. Apart from this, theoretical elucidation of the experimental outcome has also been supported by applying density functional theory (DFT) to the ligand and the complex. In situ, the solution of L2 + Hg2+ complex displayed high reversibility by I− through Hg2+ displacement approach. This reversibility in fluorescence suggested that the promising applicability of chemosensor as “off–on–off” naked eye sensor and practical applicability of L2 as an INHIBIT logic gate based on the emission changes with the inputs of Hg2+ and I− was investigated.

Peptide YY (3–36) modulates intracellular calcium through activation of the phosphatidylinositol pathway in hippocampal neurons

Peptide YY (PYY) belongs to the neuropeptide Y (NPY) family, which also includes the pancreatic polypeptide (PP) and NPY. PYY is secreted by the intestinal L cells, being present in the blood stream in two active forms capable of crossing the blood brain barrier, PYY (1–36) and its cleavage product, PYY (3–36). PYY is a selective agonist for the Y2 receptor (Y2R) and these receptors are abundant in the hippocampus. Here we investigated the mechanisms by which PYY (3–36) regulates intracellular Ca2+ concentrations ([Ca2+]i) in hippocampal neurons by employing a calcium imaging technique in hippocampal cultures. Alterations in [Ca2+]i were detected by changes in the Fluo-4 AM reagent emission. PYY (3–36) significantly increased [Ca2+] from the concentration of 10−11M as compared to the controls (infusion of HEPES-buffered solution (HBS) solution alone). The PYY (3–36)-increase in [Ca2+]i remained unchanged even in Ca2+-free extracellular solutions. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pump (SERCA pump) inhibition partially prevent the PYY (3–36)-increase of [Ca2+]i and inositol 1,4,5-triphosphate receptor (IP3R) inhibition also decreased the PYY (3–36)-increase of [Ca2+]i. Taken together, our data strongly suggest that PYY (3–36) mobilizes calcium from the neuronal endoplasmic reticulum (ER) stores towards the cytoplasm. Next, we showed that PYY (3–36) inhibited high K+-induced increases of [Ca2+]i, suggesting that PYY (3–36) could also act by activating G-protein coupled inwardly rectifying potassium K+ channels. Finally, the co-infusion of the Y2 receptor (Y2R) antagonist BIIE0246 with PYY (3–36) abolished the [Ca2+]i increase induced by the peptide, suggesting that PYY (3–36)-induced [Ca2+]i increase in hippocampal neurons occurs via Y2Rs.

Benzothiazole-Based Fluorescent Sensor for Ratiometric Detection of Zn(II) Ions and Secondary Sensing PPi and Its Applications for Biological Imaging and PPase Catalysis Assays

In this paper, we designed and synthesized three benzothiazole-based fluorescent probes L1, L2, and L3 for zinc ion detection. Among various metal ions, only the zinc ion exhibited fluorescence enhancement at 475 nm accompanied by the blue-shift emission wavelength in HEPES buffer solution containing probes. Through titration experiment, the detection limit of L1 for zinc ion sensing was calculated to be as low as 7 nM, which showed a high sensitivity. Furthermore, the confocal laser scanning micrographs of HeLa cells demonstrate good cell permeability of probe L1 and selective detection of zinc ion in living cells. The L1–Zn2+ complex was further used for pyrophosphate (PPi) sensing in HEPES buffer solution, the limit of detection was calculated to be as low as 60 nM. L1–Zn2+ can monitor the enzyme catalyzed degradation process of PPi, thus providing a meaningful way for tracking of zinc ion and pyrophosphate in biological systems.

CA2+ SIGNALLING IN HUMAN BRAIN PERICYTES: INVESTIGATION OF EFFECTS OF AMYLOID BETA TREATMENT

Pericytes are contractile cells that regulate blood flow in the microvasculature. In the central nervous system (CNS), they are an important component of the gliovascular unit, a multicomponent cellular structure that is impacted by amyloid pathology. Here we test the effect of a synthetic human amyloid beta 1:42 peptide (hAβ 1–42) on resting Ca2+ concentrations and G-protein coupled receptor (GPCR)-mediated Ca2+ responses in human CNS pericytes. Human CNS pericytes and cell culture media were purchased from sciencellonline. Cells were grown in flasks according to the supplier's protocols. For Ca2+ imaging, cells were plated onto poly-D-lysine coated glass coverslips and pre-incubated for ∼24 hours with culture medium containing hAβ 1–42 (500 nM) or vehicle (DMSO 0.05%). The pericytes were then loaded with the Ca2+-sensitive ratiometric dye fura-2, before being placed on an inverted microscope equipped for single cell microfluorimetry. Imaging was performed in a standard HEPES-buffered saline solution (HBSS). HBSS variants containing three concentrations of glucose, 0.1, 2.5 and 6 mM, were individually examined. Pericytes exposed to hAβ1–42 displayed a lower basal Ca2+ concentration when experiments were performed in 2.5 and 6 mM glucose, although this was not observed in 0.1 mM glucose. We also tested Ca2+ responses to extracellular applications of a number of GPCR agonists relevant to pericyte function. Robust Ca2+ responses to endothelin1, bradykinin and histamine were noted; the latter producing the biggest overall population responses. Quite similar agonist responses were seen in bath solutions containing the different glucose concentrations. Furthermore, hAβ1–42 treatment produced no notable effect on agonist-induced Ca2+ responses. In summary, our initial data indicate that short term exposure of pericytes to hAβ1–42 can lower resting Ca2+ but has no discernible effect on GPCR-evoked pericyte Ca2+ signalling.

Indirect Approach for CN– Detection: Development of “Naked-Eye” Hg2+-Induced Turn-Off Fluorescence and Turn-On Cyanide Sensing by the Hg2+ Displacement Approach

The coumarin-thiol-based colorimetric and fluorimetric chemosensor (E)-7-(diethylamino)-3-((2-mercaptophenylimino)methyl)-2H-chromen-2-one 1a was designed, synthesized, and evaluated for anion detection through the metal displacement mechanism. Chemosensor 1a exhibited a high sensitivity and selectivity to Hg2+ ion detection in HEPES-buffered solution [20 mM, 3:7 CH3CN/H2O (v/v), pH 7.0] with a 1:1 binding stoichiometry and a 5.549 × 105 M–1 binding constant. The fluorescence intensity of chemosensor 1a was selectively quenched by Hg2+, and an obvious color change from yellowish green to orange was observed by the naked eye. Moreover, a solution of the in situ generated 1a + Hg2+ complex displayed a high selectivity to CN– through the Hg2+ displacement approach. The gradual addition of CN– to the resultant solution of the 1a + Hg2+ complex induced its fluorescence recovery with a detection limit of 31 nM. This reversibility in fluorescence suggests the promising applicability of chemosensor 1a as an “on–o...

Synthesis and evaluation of a new fluorescein and rhodamine B-based chemosensor for highly sensitive and selective detection of cysteine over other amino acids and its application in living cell imaging

A new fluorescent probe (TRDM-F) based on a molecular chain of open-loop maleic anhydride, rhodamine B and fluorescein was designed, synthesized and evaluated to develop a chemosensor. Photochemical studies show that fluorescence properties of TRDM-F are dominated by fluorescein group in the pH range of 2–10. UV–vis absorption and fluorescence spectroscopic studies further display a high selectivity towards Cys over GSH/Hcy and other amino acids and a high sensitivity towards Cys (0.088–270μM) with 95-fold fluorescence enhancement in a HEPES buffer solution (10mM, pH=7.4) containing 20% MeCN at room temperature. We further demonstrated that the proposed method was satisfactorily applied to the discrimination of Cys in living Hela cells with low cell toxicity.

Study of Efficiency of Coupling Peptides with Gold Nanoparticles

Abstract Fluorescence spectroscopy was used to investigate the efficiency of coupling peptides to gold nanoparticles via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS). In this study, the effects of experiment conditions including the buffer solution (PBS, HEPES, Tris-HCl, and borate buffer solution), pH (6.5–9.0) and concentrations of buffer solution (10, 25, 40 and 50 mM), concentrations of NHS and EDC (from 0.2 M NHS-0.01 M EDC to 1.0 M NHS-0.5 M EDC), ratios of NHS to EDC (0, 0.5, 1.0, 2.0 and 2.5), and the coupling reaction time (4, 8, 12, 24 and 36 h) on the coupling efficiency were investigated and the optimized conditions were developed. These results indicated that the optimal experimental conditions were: pH 7.0 and 25 mM of the HEPES buffer solution, the concentrations ratios of NHS to EDC of 2:1 (0.4 M NHS-0.2 M EDC), and coupling reaction time of 24 h.

Rapid Detection of pM Concentration of Insulin Using Microwave Whispering Gallery Mode

In this paper, we are going to investigate insulin, a bio-molecule responsible for the onset of diabetes, which is generally diagnosed using the conventional ELISA method. Its picomolar concentration in blood and short life time was a challenge in developing a sensor sensitive to such small volumes. This paper presents an experimental study of the dielectric properties of hepes buffer solution with varying concentration of insulin, a peptide hormone at microwave frequency range. A composite whispering gallery electric mode ( $WGE_{800}$ ) microwave dielectric resonator with sapphire and polycarbonate disposable sample holding disc has been used at 17.59 GHz. From observations, imaginary part of permittivity has been found very sensitive to changes in insulin concentration ranging from $2.0327\times 10^{-8} \% $ to $21.9995\times 10^{-8} \% $ . Studies show that the present method has the potential to detect and sense ultralow concentration of bioanalyte with regression coefficient 0.943 and sensitivity of 0.039 for 10 pM change in insulin concentration.

Indigo Carmine-Cu complex probe exhibiting dual colorimetric/fluorimetric sensing for selective determination of mono hydrogen phosphate ion and its logic behavior

A new selective probe based on copper complex of Indigo Carmine (IC-Cu2) for colorimetric, naked-eye, and fluorimetric recognition of mono hydrogen phosphate (MHP) ion in H2O/DMSO (4:1v/v, 1.0mmolL−1 HEPES buffer solution pH7.5) was developed. Detection limit of HPO42− determination, achieved by fluorimetric and 3lorimetric method, are 0.071 and 1.46μmolL−1, respectively. Potential, therefore is clearly available in IC-Cu2 complex to detect HPO42− in micromolar range via dual visible color change and fluorescence response. Present method shows high selectivity toward HPO42− over other phosphate species and other anions and was successfully utilized for analysis of P2O5 content of a fertilizer sample. The results obtained by proposed chemosensor presented good agreement with those obtained the colorimetric reference method. INHIBIT and IMPLICATION logic gates operating at molecular level have been achieved using Cu2+and HPO42− as chemical inputs and UV–Vis absorbance signal as output.

Synthesis of Rhodamine-Based Chemosensor for Fe3+ Selective Detection with off-on Mechanism and its Biological Application in DL-Tumor Cells

A novel rhodamine-based chemosensor (R1) has been designed and synthesised for identifying Fe3+ ions among different metal ions in aqueous medium. The sensitivity test has been carried out by using UV-vis and fluorescence methods taking μM concentration of R1 in HEPES buffer solution and the complexation has been described through fluorescence ‘off-on’ mechanism. Metallation of R1 was found to be tolerant with respect to tumor cells proliferation and viability of human peripheral blood mononuclear cells and RBC. It has also been demonstrated that the significant uptake of the chemosensor by the tumor cells was observed in cell imaging analysis.

“Cascade recognition” of Cu2+ and H2PO4− with high sensitivity and selectivity in aqueous media based on the effect of ESIPT

Organic molecular sensors have the advantage of being used through easy, fast, economical and reliable optical methods for detecting toxic metal ions in the environment. In the present work, a simple but specific organic ligand N,N-(3,3′-dimethyl biphenyl)-bis(4-diethylaminosalicyladimine) (Q2) was designed and synthesized according to the excited state intramolecular proton transfer (ESIPT). Q2 can be regarded as a colorimetric and fluorescent sensor for relay recognition of Cu2+ and H2PO4− in DMSO/H2O (8:2, v/v) HEPES buffer solutions. Moreover, sensor Q2 showed a highly sensitive and selective response towards Cu2+ and H2PO4− over other metal ions and anions. Particularly, the fluorescence “ON–OFF–ON” responding circle could be repeated over 4 times by the sequential addition of Cu2+ and H2PO4−. Notably, the actual usage of sensor Q2 was further demonstrated by test kits and silica gel plates. The fluorescence changes of Q2 upon the addition of Cu2+ and H2PO4− were utilized as an INHIBIT logic gate at the molecular level, using Cu2+ and H2PO4− as chemical inputs and the fluorescence intensity signal as the output.

A highly sensitive and selective sensor based on imidazo[1,2- a ]pyridine for Al 3+

Abstract A new fluorescent sensor, N′ -[(2-hydroxyphenyl)methylidene]imidazo[1,2- a ]pyridine-2-carbohydrazide ( S ), has been synthesized via Schiff-base condensation between imidazo[1,2- a ]pyridine-2-carbohydrazide and salicylaldehyde. It exhibited high sensitivity and selectivity towards Al 3+ through a significant “turn-on” fluorescence response at 446 nm in DMSO-H 2 O (9:1,v:v) 10 mM of HEPES buffer solution at pH 7.4 with a detection limit down to 4.55 × 10 −11 M. Intriguingly, sensor S could also act as a pH sensor to detect the pH values of alkali environment by inhibiting the PET process. The optimized structure of S and its sensing mechanism for Al 3+ has been confirmed by density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations with 6-31G(d) basis sets using a suite of Gaussian 09 programs.

Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells.

Genetic modification (“transfection”) of mammalian cells using non-viral, synthetic agents such as polycations, is still a challenge. Polyplex formation between the DNA and the polycation is a decisive step in such experiments. Star-shaped polycations have been proposed as superior transfection agents, yet have never before been compared side-by-side, e.g., in view of structural effects. Herein four star-shaped polycationic structures, all based on (2-dimethylamino) ethyl methacrylate (DMAEMA) building blocks, were investigated for their potential to deliver DNA to adherent (CHO, L929, HEK-293) and non-adherent (Jurkat, primary human T lymphocytes) mammalian cells. The investigated vectors included three structures where the PDMAEMA arms (different arm length and grafting densities) had been grown from a center silsesquioxane or silica-coated γ-Fe2O3-core and one micellar structure self-assembled from poly(1,2-butadiene)-block PDMAEMA polymers. All nano-stars combined high transfection potential with excellent biocompatibility. The micelles slightly outperformed the covalently linked agents. For method development and optimization, the absolute amount of polycation added to the cells was more important than the N/P-ratio (ratio between polycation nitrogen and DNA phosphate), provided a lower limit was passed and enough polycation was present to overcompensate the negative charge of the plasmid DNA. Finally, the matrix (NaCl vs. HEPES-buffered glucose solution), but also the concentrations adjusted during polyplex formation, affected the results.

1,2′]Biindenylidene-3,1′-3′-trion(bindone): Colorimetric detection of volatile organic compounds(VOCs) ethylamine using highly selective Hg2+ chemosensor in aqueous solution

A colorimetric chemosensor based on [1,2′]biindenylidene-3,1′-3′-trion(bindone) ion (BD) has been investigated for high selectivity and sensitivity toward Hg2+ in HEPES buffer solution. Interestingly, the colorless solution of the BD-Hg2+ complex could be recovered after addition of VOC EtNH2. Sensing mechanism was confirmed by 1H NMR studies. We have investigated ability of complex formation based on the association constant, Kass; the binding ability for the complex formation of BD-Hg2+ and EtNH2 is greater (Kass = 8.6 × 104 M−1) than that of the BD with Hg2+ (Kass = 8.0 × 103 M−1). EtNH2 gas test strip was made by adsorbing BD-Hg2+ on to the test paper. The test strip was exposed to EtNH2 gas. And the resulting color changing properties were clearly demonstrated.

Highly Selective Ratiometric Fluorescent Probes for Detection of Perborate Based on Excited‐State Intramolecular Proton Transfer (ESIPT) in Environmental Samples and Living Cells

AbstractBenzthiazole‐based ratiometric fluorescent probes acetylphenylbenzthiazole (APBT) and acetylnaphthylbenzthiazole (ANBT), were synthesized and characterized. These two excited‐state intramolecular proton transfer (ESIPT)‐based first ratiometric fluorescent probes were shown to be selective and sensitive for perborate (BO3–) among the 15 anions studied in HEPES buffer medium by fluorescence, absorption, and visual emission color change. Upon treatment with perborate in aqueous HEPES buffer solution, the acetyl protective group of probes APBT and ANBT was removed readily and ESIPT of the probes was switched on, which resulted in remarkable photo‐physical changes. Time dependent‐ density functional theory (TD‐DFT) calculations were performed in order to demonstrate the electronic properties of APBT and ANBT and their anion. An easy‐to‐prepare test paper, obtained by dipping the paper into the solution of APBT, was able to detect BO3– in practical samples. Moreover, the utility of the APBT and ANBT probes in perborate recognition has also been demonstrated in Vero cells using Confocal imaging.

A selective fluorescent probe for the detection of Cd(2+) in different buffer solutions and water.

A simple fluorescent probe NHQ based on quinoline was successfully prepared via one-step synthesis. The probe NHQ exhibited "turn-on" fluorescence and excellent selectivity toward Cd(2+) in different buffer solutions such as Tris-HCl buffer solution, HEPES buffer solution, and PBS buffer solution, and even in water. Moreover, the binding model of NHQ with Cd(2+) was definitely confirmed by the single crystal X-ray diffraction studies of the complex.

The Effect and Underlying Mechanism of Ethanol on Intracellular H(+) -Equivalent Membrane Transporters in Human Aorta Smooth Muscle Cells.

The presence of intracellular pH (pHi ) regulators, including Na(+) -H(+) exchanger (NHE), Na(+) -HCO3- co-transporter (NBC), Cl(-) /OH(-) exchanger (CHE), and Cl(-) /HCO3- exchanger (AE), have been confirmed in many mammalian cells. Alcohol consumption is associated with increased risk of cardiovascular disorder. The aims of the study were to identify the possible transmembrane pHi regulators and to explore the effects of ethanol (EtOH) (10 to 300mM) on the resting pHi and pHi regulators in human aorta smooth muscle cells (HASMCs). HASMCs were obtained from patients undergoing heart transplant. The pHi was measured by microspectrofluorimetry with the pH-sensitive dye, BCECF-AM. The following results are obtained. (i) In cultured HASMCs, the resting pHi was 7.19±0.04 and 7.13±0.02 for HEPES- and CO2 /HCO3--buffered solution, respectively. (ii) Two different Na(+) -dependent acid-equivalent extruders, including NHE and Na(+) -coupled HCO3- transporter, functionally coexisted. (iii) Two different Cl(-) -dependent acid loaders (CHE and AE) were functionally identified. (iv) EtOH induced a biphasic, concentration-dependent change in resting pHi (+0.25pH unit at 100mM but only +0.05 pH unit at 300mM) in bicarbonate-buffered solution, while caused a concentration-dependent decrease in resting pHi (-0.06 pH unit at 300mM) in HEPES-buffered solution. (v) The effect of EtOH on NHE activity was also biphasic: increase of 40% at lower concentration of 10mM, followed by decrease of 30% at higher concentration of 300mM. (vi) The increase in Na(+) -coupled HCO3- transporter activity by EtOH was concentration dependent. (vii) The effect of EtOH on CHE and AE activities was both biphasic: increase of ~25% at 30mM, followed by decrease of 10 to 25% at 100mM, and finally increase of 15 to 20% at 300mM. This study demonstrated that 2 acid extruders and 2 acid loaders coexisted functionally in HASMCs and that EtOH induced a biphasic, concentration-dependent change in resting pHi by altering the activity of the 2 acid extruders, NHE and Na(+) -coupled HCO3- transporter, and the 2 acid loaders, CHE and AE.

A Novel Fluorescent Optode for Recognition of Zn(2+) ion Based on N,N(')-bis-(1-Hydroxypheylimine)2,5-Thiophenedicarobxaldehyde (HPTD) Schiff Base.

A novel fluorescent sensing film has been proposed for sensitive determination of Zn(2+) ion in EtOH-HEPES buffer solution (20 mM HEPES, 50:50, v/v, pH 7.0). The novel sensor based on incorporating a novel qaudridentate Schiff base N,N(')-bis-(1-hydroxypheylimine)2,5-thiophenedicarobxaldehyde (HPTD) as ionophore in the plasticized PVC membrane containing bis(2-ethylhexyl)sebecate (DOS) as plasticizer. The novel optode membrane works on the basis of a cation-exchange mechanism and shows a significant fluorescent emission enhancement on exposure to HEPES buffer solution of pH7.0 containing Zn(2+) ion. Under optimal conditions, the proposed sensing film displays a linear range of 1.0×10(-12) to 8.6×10(-4) M with a limit of detection 5.3×10(-13) M. The response characteristics of the sensor including reversibility, reproducibility, response time and lifetime are discussed in detail. The optode membrane has been applied to determine Zn(2+) in various real water samples.

Myocyte contractility can be maintained by storing cells with the myosin ATPase inhibitor 2,3 butanedione monoxime.

Isolated intact myocytes can be used to investigate contractile mechanisms and to screen new therapeutic compounds. These experiments typically require euthanizing an animal and isolating fresh cells each day or analyzing cultured myocytes, which quickly lose their rod-shaped morphology. Recent data suggest that the viability of canine myocytes can be prolonged using low temperature and N-benzyl-p-toluene sulfonamide (an inhibitor of skeletal myosin ATPase). We performed similar studies in rat myocytes in order to test whether the cardiac myosin ATPase inhibitors 2,3-Butanedione monoxime (BDM) and blebbistatin help to maintain cell-level function over multiple days. Myocytes were isolated from rats and separated into batches that were stored at 4°C in a HEPES-buffered solution that contained 0.5 mmol L−1 Ca2+ and (1) no myosin ATPase inhibitors; (2) 10 mmol L−1 BDM; or (3) 3 μmol L−1 blebbistatin. Functional viability of myocytes was assessed up to 3 days after the isolation by measuring calcium transients and unloaded shortening profiles induced by electrical stimuli in inhibitor-free Tyrode's solution. Cells stored without myosin ATPase inhibitors had altered morphology (fewer rod-shaped cells, shorter diastolic sarcomere lengths, and membrane blebbing) and were not viable for contractile assays after 24 h. Cells stored in BDM maintained morphology and contractile function for 48 h. Storage in blebbistatin maintained cell morphology for 72 h but inhibited contractility. These data show that storing cells with myosin ATPase inhibitors can extend the viability of myocytes that will be used for functional assays. This may help to refine and reduce the use of animals in experiments.

High effective cytosolic H+ buffering in mouse cortical astrocytes attributable to fast bicarbonate transport.

Cytosolic H(+) buffering plays a major role for shaping intracellular H(+) shifts and hence for the availability of H(+) for biochemical reactions and acid/base-coupled transport processes. H(+) buffering is one of the prime means to protect the cell from large acid/base shifts. We have used the H(+) indicator dye BCECF and confocal microscopy to monitor the cytosolic H(+) concentration, [H(+)]i, in cultured cortical astrocytes of wild-type mice and of mice deficient in sodium/bicarbonate cotransporter NBCe1 (NBCe1-KO) or in carbonic anhydrase isoform II (CAII-KO). The steady-state buffer strength was calculated from the amplitude of [H(+)]i transients as evoked by CO2/HCO3(-) and by butyric acid in the presence and absence of CO2/HCO3(-). We tested the hypotheses if, in addition to instantaneous physicochemical H(+) buffering, rapid acid/base transport across the cell membrane contributes to the total, "effective" cytosolic H(+) buffering. In the presence of 5% CO2/26 mM HCO3(-), H(+) buffer strength in astrocytes was increased 4-6 fold, as compared with that in non-bicarbonate, HEPES-buffered solution, which was largely attributable to fast HCO3 (-) transport into the cells via NBCe1, supported by CAII activity. Our results show that within the time frame of determining physiological H(+) buffering in cells, fast transport and equilibration of CO2/H(+)/HCO3(-) can make a major contribution to the total "effective" H(+) buffer strength. Thus, "effective" cellular H(+) buffering is, to a large extent, attributable to membrane transport of base equivalents rather than a purely passive physicochemical process, and can be much larger than reported so far. Not only physicochemical H(+) buffering, but also rapid import of HCO3(-) via the electrogenic sodium-bicarbonate cotransporter NBCe1, supported by carbonic anhydrase II (CA II), was identified to enhance cytosolic H(+) buffer strength substantially.