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PubMed2000

Mechanisms of endothelial cell swelling from lactacidosis studied in vitro.

One of the early sequelae of ischemia is an increase of circulating lactic acid that occurs in response to anaerobic metabolism. The purpose of the present study was to investigate whether lactic acidosis can induce endothelial swelling in vitro under closely controlled extracellular conditions. Cell volume of suspended cultured bovine aortic endothelial cells was measured by use of an advanced Coulter technique employing the "pulse area analysis" signal-processing technique (CASY1). The isosmotic reduction of pH from 7.4 to 6.8 had no effect on cell volume. Lowering of pH to 6.6, 6.4, or 6.0, however, led to significant, pH-dependent increases of cell volume. Swelling was more pronounced in bicarbonate-buffered media than in HEPES buffer. Specific inhibition of Na(+)/H(+) exchange by ethylisopropylamiloride completely prevented swelling in HEPES-buffered media. Pretreatment with ouabain to partially depolarize the cells did not affect the degree of acidosis-induced swelling. In bicarbonate-buffered media, the inhibition of transmembrane HCO(3)(-) transport by DIDS reduced swelling to a level comparable with that seen in the absence of bicarbonate ions. Lactacidosis-induced endothelial swelling, therefore, is a result of intracellular pH regulatory mechanisms, namely, Na(+)/H(+) exchange and bicarbonate-transporting carriers.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Chloride transport by rat renal proximal tubule: effects of bicarbonate absorption.

In the early part of the proximal tubule chloride concentration is increased above that in plasma, a change attributed to preferential absorption of bicarbonate with sodium and water. To determine the mechanism(s) of the preferential absorption of HCO3 over chloride, in vivo paired perfusions were done in surface proximal tubules of the rat kidney. Each tubule was perfused with a control bicarbonate solution, similar in ionic composition to the solution normally present in the early part of the proximal tubule, and one of the experimental solutions that were modified by either replacing all of bicarbonate with sulfate, N-2-hydroxyethylpiperazine-N'-ethanesulfonate (HEPES), or all bicarbonate with sulfate and sodium with lithium, or addition of acetazolamide (ATZ), or cyanide (CN). Sufficient mannitol was added to reduce net fluid flux to zero. When sulfate replaced bicarbonate in luminal fluid, net chloride absorption was enhanced, a change associated with low lumen HCO3 and low pH. ATZ inhibited HCO3 absorption, whereas net Cl absorption increased, an effect associated with high HCO3 and low pH. With HEPES-plus-ATZ solutions, chloride absorption also increased but, in contrast to SO4 or ATZ solutions, pH increased. With sulfate and lithium replacement chloride absorption also increased despite net sodium secretion. CN, a metabolic inhibitor, inhibited HCO3 and sodium absorption but stimulated net chloride absorption. The data indicate that HCO3 absorption inhibits chloride absorption, a process independent of luminal HCO3 per se, luminal pH, sodium absorption, and transepithelial volume flux and voltage.

Absorption↗
PubMed1986

Stimulation of chloride transport by HCO3-CO2 in rabbit cortical collecting tubule.

We examined both the role of HCO3-CO2 in Cl transport as well as the effect of in vivo acid-base status on Cl transport by the rabbit cortical collecting tubule. The lumen-to-bath 36Cl tracer flux, expressed as the rate coefficient KCl, was measured in either HEPES-buffered (CO2-free) or HCO3-CO2-containing solutions. Amiloride was added to the perfusate to minimize the transepithelial voltage and thus the electrical driving force for Cl diffusion. Because KCl fell spontaneously with time in HCO3-CO2 solutions in the absence but not the presence of cAMP, we used cAMP throughout to avoid time-dependent changes. Acute in vitro removal of bath HCO3-CO2 reduced KCl. Acetazolamide addition in HEPES-buffered solutions also lowered KCl; KCl could be restored to control values by adding exogenous HCO3-CO2 in the presence of acetazolamide. In vivo acid-base effects on Cl transport were determined by dissecting tubules from either NaHCO3-loaded or NH4Cl-loaded rabbits. Tubules from HCO3-loaded rabbits had higher rates of Cl self exchange. Acute in vitro addition of bath HCO3-CO2 increased KCl and did so to a greater degree in tubules from HCO3-loaded rabbits. Most of this effect of HCO3-CO2 addition on KCl could not be accounted for by Cl-HCO3 exchange; rather, it appeared due to stimulation of Cl self exchange. The data are consistent with 36Cl transport occurring via Cl-HCO3 exchange as well as Cl self exchange. Both processes are acutely stimulated by HCO3 and/or Co2, and both are chronically regulated by in vivo acid-base status.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

HCO3 accumulation in proximal tubule: roles of carbonic anhydrase, luminal buffers, and pH.

Most of filtered bicarbonate is reabsorbed in the early proximal tubule, and the high blood-to-lumen HCO3 concentration gradient generated is then maintained in the distal proximal tubule. To determine the factor(s) that prevent reaccumulation of HCO3 in the lumen, surface proximal tubules of the rat kidney were perfused in vivo. All perfusion solutions were similar in ionic composition to late proximal tubule fluid but, instead of HCO3, contained sulfate (SO4) or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Bicarbonate concentration ([HCO3]L) was measured by microcalorimetry, while collected fluid PCO2 was maintained in vitro at either renal cortical or atmospheric levels. A new single microelectrode was used to simultaneously measure PCO2 and luminal pH (pHL). With SO4 solution, pHL was 6.84 +/- 0.06, PCO2 was 50.8 +/- 5.0 mmHg, and [HCO3]L was 8.7 +/- 0.9 mM. When 10(-3) M acetazolamide (ATZ) was added to the perfusate, pHL was 7.00 +/- 0.08, PCO2 remained unchanged, [HCO3]L was 12.1 +/- 1.3 mM, and the rate of HCO3 accumulation increased by approximately 50%. When SO4 was replaced with HEPES, pHL increased to 7.18 +/- 0.05, PCO2 was unchanged, [HCO3]L was 16.5 +/- 1.4 mM, and the rate of HCO3 accumulation doubled. Because measured [HCO3]L and that calculated from pHL and PCO2 were approximately the same, the net gain of HCO3 occurred in vivo, a result of either HCO3 backflux and/or intraluminal generation from CO2. By allowing the collected fluid PCO2 to fall to near zero prior to [HCO3]L measurement, we estimate that approximately 50% of the total gain of HCO3 with all solutions was due to backflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effects of intracellular pH on hypoxic vasoconstriction in rat lungs.

Isolated rat lungs perfused with physiological salt-Ficoll solutions were studied to test whether hypoxic pulmonary vasoconstriction was potentiated by increases in intracellular pH (pHi) and blunted by decreases in pHi. Whereas addition to perfusate of 5 nM phorbol myristate acetate (PMA), a stimulator of exchange of intracellular H+ for extracellular Na+, potentiated hypoxic vasoconstriction, 1 mM amiloride, an inhibitor of Na+-H+ exchange, blunted the hypoxic response. Hypoxic vasoconstriction was also potentiated by the weak bases NH4Cl (20 mM), methylamine (10 mM), and imidazole (5 mM) and was inhibited by the weak acid sodium acetate (40 mM). NH4Cl, imidazole, and acetate had the same effects on KCl-induced vasoconstriction and on the hypoxic response. Hypoxic vasoconstriction was greater in lungs perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution than in those perfused with CO2/HCO3--buffered solution. Similarly, lungs perfused with CO2/HCO3--buffered solution containing 1.8 mM Cl- (NaNO3 and KNO3 substituted for NaCl and KCl) had larger hypoxic and angiotensin II pressor responses than those perfused with 122.5 mM Cl-. Because PMA, NH4Cl, methylamine, imidazole, HEPES-buffered solutions, and low-Cl- solutions can cause increases in pHi and amiloride and acetate can cause decreases in pHi, these results suggest that intracellular alkalosis and acidosis, respectively, potentiate and blunt vasoconstrictor responses to hypoxia and other stimuli in isolated rat lungs. These effects could be related to pHi-dependent changes in either the sensitivity of the arterial smooth muscle contractile machinery to Ca2+ or the release of a vasoactive mediator or modulator by some other lung cell.

Acetates↗
PubMed2000

Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway.

Previous studies have suggested that voltage-gated Ca(2+) influx in glomus cells plays a critical role in sensory transduction at the carotid body chemoreceptors. The purpose of the present study was to determine the effects of hypoxia on the Ca(2+) current in glomus cells and to elucidate the underlying mechanism(s). Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca(2+) current was monitored using the whole cell configuration of the patch-clamp technique, with Ba(2+) as the charge carrier. Hypoxia (pO(2) = 40 mmHg) augmented the Ca(2+) current by 24 +/- 3% (n = 42, at 0 mV) in a voltage-independent manner. This effect was seen in a CO(2)/HCO(3)(-)-, but not in a HEPES-buffered extracellular solution at pH 7.4 (n = 6). When the pH of a HEPES-buffered extracellular solution was lowered from 7.4 to 7. 0, hypoxia augmented the Ca(2+) current by 20 +/- 5% (n = 4, at 0 mV). Nisoldipine, an L-type Ca(2+) channel blocker (2 microM, n = 6), prevented, whereas, omega-conotoxin MVIIC (2 microM, n = 6), an inhibitor of N and P/Q type Ca(2+) channels, did not prevent augmentation of the Ca(2+) current by hypoxia, implying that low oxygen affects L-type Ca(2+) channels in glomus cells. Protein kinase C (PKC) inhibitors, staurosporine (100 nM, n = 6) and bisindolylmaleimide (2 microM, n = 8, at 0 mV), prevented, whereas, a protein kinase A inhibitor (4 nM PKAi, n = 10) did not prevent the hypoxia-induced increase of the Ca(2+) current. Phorbol 12-myristate 13-acetate (PMA, 100 nM), a PKC activator, augmented the Ca(2+) current by 20 +/- 3% (n = 8, at 0 mV). In glomus cells treated with PMA overnight (100 nM), hypoxia did not augment the Ca(2+) current (-3 + 4%, n = 5, at 0 mV). Immunocytochemical analysis revealed PKCdelta-like immunoreactivity in the cytosol of the glomus cells. Following hypoxia (6% O(2) for 5 min), PKCdelta-like immunoreactivity translocated to the plasma membrane in 87 +/- 3% of the cells, indicating PKC activation. These results demonstrate that hypoxia augments Ca(2+) current through L-type Ca(2+) channels via a PKC-sensitive mechanism.

Animals↗
PubMed1998

The effect of intrinsic fluoride in cows' milk on in vitro enamel demineralization.

The fluoride concentration in cows' milk has been reported to vary with the fluoride levels in drinking water but it seldom exceeds 0.5 microg/ml. This raised a question as to whether any caries-protective effect could be attributed to the intrinsic fluoride of milk. Two samples of cows' milk with intrinsic fluoride concentrations of 0.03 and 0.3 microg/ml, respectively, were assessed for their protective effect on enamel in an in vitro demineralization model at relatively severe and mild acidic challenges (pH 4.6 and 5.0, respectively). Polished enamel discs were incubated individually in 5.0 ml of demineralization solution for 20 h per day alternated with 1-hour incubations in 1.0 ml of milk or control buffers: group 1, demineralization solution only (negative control); group 2, milk with 0.03 microg/ml fluoride; group 3, milk with 0.03 microg/ml fluoride; supplemented with NaF to 0.3 microg/ml fluoride; group 4, milk with 0.3 microg/ml fluoride; group 5, 0.3 microg/ml fluoride in 20 mM HEPES, pH 6.7; group 6, milk with 0.03 microg/ml fluoride supplemented with NaF to 5.0 microg/ml fluoride (positive control). The solutions were renewed each day and the calcium concentration in the demineralization solutions was followed during 4 days. The results showed that the protective effect of intrinsic milk fluoride on enamel is limited by the severity of the acidic challenge: There was a significant inhibition of the demineralization in groups 3-6 compared to groups 1 and 2, but only at pH 5.0 (p<0.0001) and not at pH 4.6 (p = 0.2). The organic components of milk had limited protection against demineralization because milk and HEPES with the same fluoride concentration gave similar results. The 36% reduction in calcium loss at pH 5.0 by treatment with milk with only 0.3 microg/ml fluoride is an indication that intrinsic milk fluoride has some caries-protective properties.

Acetates↗
PubMed1996

pH-dependent changes in interphotoreceptor matrix domains surrounding cone photoreceptors.

The cone matrix sheath is a biochemically and structurally distinct domain of the retinal interphotoreceptor matrix (IPM). In the present study, the insoluble components of the cone matrix sheath were extracted from bovine retina, and examined for pH-dependent morphological changes by light and electron microscopy. After soaking unfixed retinas in 25 mM HEPES-buffered solution (pH 2.0), sheets of aqueous-insoluble IPM containing cone matrix sheaths were physically dissociated from the inner retina. The insoluble IPM preparations were treated with 1.0% Triton X-100 to extract contaminating photoreceptor outer segment membrane, and exposed to HEPES-buffered solutions at pH 2.0, 4.5, 7.0, or 9.5. The cone matrix sheath, specifically stained with peanut agglutinin, appeared finely granular at pH 2.0, but was markedly condensed at pH 4.5. Cone sheath morphology became dispersed and appeared as a network of thick fibrils at pH 7.0, and further dispersed fibrillar and granular elements at pH 9.5. These findings suggest that pH changes in the interphotoreceptor space can alter the molecular organization of the IPM and influence the function of the IPM in the transport of nutrients and metabolites, and in retinal adhesion.

Animals↗
PubMed1986

Intra- and extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells.

External or internal application of proton on the calcium current was investigated using the whole-cell patch clamp technique in single ventricular cells of the guinea pig. The effect of acid pHo on the calcium current depended on the strength of the buffer in the internal solution. In the presence of 5 mM HEPES in the pipette, external acidification depressed calcium current with the half maximum inhibition of pHo 5.5. When the intracellular pH was buffered strongly to pH 7.2 by 50 mM HEPES within the pipette solution, half maximum inhibition of the calcium current was shifted to the acidic side markedly. When acid was applied in the patch electrode, calcium current was hardly affected by the pH values down to 6.0 in the pipette solution but was depressed by approximately half on further acidification to pH 4.5. When the Na+-H+ exchange system was blocked by superfusing the cell with either amiloride or Na+-deficient solutions (Tris substituted), calcium current was decreased by half at a pipette pH of around 6.5 and was completely and irreversibly blocked at pHpip 6.0. From the above results, we concluded that the calcium current of the ventricular cell is much more sensitive to intracellular protons than extracellular ones.

Animals↗
PubMed1994

DIDS-sensitive pHi regulation in single rat cardiac myocytes in nominally HCO3-free conditions.

The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi in the spontaneously hypertensive rat (SHR) and in normal rat cardiac myocytes under nominally HCO3-free (20 mmol/L HEPES-buffered) conditions. When only the Na-H exchanger was blocked, the intrinsic buffering power (beta i) in SHR myocytes was significantly higher than when both the Na-H exchanger and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pHi regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. Similar low values for beta i were also found for normal rat myocytes in Na(+)-free conditions. In Cl(-)-free solution under nominally HCO3-free conditions, in both normal and SHR myocytes, the pHi slowly alkalinized (by 0.16 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively); this alkalinization was also DIDS sensitive. The reacidification during NH4+ perfusion was inhibited 30.2 +/- 7.4% by DIDS. In addition, in the nominal absence of HCO3-, 100 mumol/L ATP acidified the pHi in both normal and SHR myocytes (by 0.21 +/- 0.03 and 0.33 +/- 0.03 pH units, respectively); this acidification was totally inhibited by 0.1 mmol/L DIDS. It has been shown, in rat cardiac myocytes, that ATP acidifies the pHi by 0.35 pH unit via stimulation of a DIDS-sensitive Cl-HCO3 exchanger in HCO3-containing solutions. Finally, we have shown, in normal cardiac myocytes, that two potent Na-H exchanger blockers, N-5-ethylisopropyl amiloride (EIPA) and N-5-methyl-N-isobutyl amiloride (MIA), only partially inhibited the pHi recovery from internal acidosis under nominally bicarbonate-free conditions. When DIDS was added at the same time as EIPA, pHi recovery from an internal acid loading was completely inhibited. Our results clearly demonstrate that in both normal and SHR cardiac myocytes, bicarbonate-dependent pHi regulators can be significantly activated under resting or acidified pHi in HEPES-buffered medium, probably because of the cellular production of CO2. The contribution of these bicarbonate-dependent pHi regulators, ie, the Na-HCO3 cotransporter and the Cl-HCO3 exchanger, cannot therefore be ignored even under nominally HCO3-free conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

Hypoxia-inducible transcription factor-1alpha promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway.

BACKGROUND: Hypoxia-inducible transcription factor-1alpha (HIF-1alpha), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a "master" gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1alpha on apoptosis by modulating HIF-1alpha gene expression in A549 cells through both siRNA knock-down and over-expression. METHODS: A549 cells were transfected with a HIF-1alpha siRNA plasmid or a HIF-1alpha expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) (5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. RESULTS: Knocking down expression of HIF-1alpha inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1alpha accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1alpha on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1alpha over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. CONCLUSION: During hypoxia in A549 cells, HIF-1alpha promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis.

Apoptosis↗
PubMed1995

Mechanisms of intracellular pH regulation during postischemic reperfusion of diabetic rat hearts.

A marked decrease in the activity of the amiloride-sensitive Na+/H+ exchanger has been demonstrated in hearts from streptozotocin (STZ)-induced diabetic rats. The aim of this study was to investigate the contribution of other specific sarcolemmal transport mechanisms to intracellular pH (pHi) recovery upon reperfusion in STZ-induced diabetic rat hearts and their relation to recovery of ventricular function. Isovolumic rat hearts were submitted to a zero-flow ischemic period of 28 min at 37 degrees C and then reperfused for 28 min. The time course of pHi decline during ischemia and of recovery on reperfusion was followed by means of 31P-labeled NMR. The perfusion buffers used were either HEPES or CO2/HCO3-. An HCO3(-)-dependent (amiloride-insensitive) mechanism contributed to pHi recovery after ischemia in the diabetic rat hearts. Even when the Na+/H+ exchanger was blocked by amiloride in nominally HCO3(-)-free solution, a rapid rise in pHi occurred during the first 3 min of reperfusion. The early rise in pHi was reduced by external lactate and inhibited by alpha-cyano-4-hydroxycinnamate. This suggested that a coupled H(+)-lactate efflux may be a major mechanism for acid extrusion in the initial stage of reperfusion. The observation of a higher functional recovery on reperfusion in diabetic hearts is in accordance with previous studies using HCO3- buffer. However, this study shows that a good recovery of function occurred even more rapidly in diabetic hearts receiving HEPES-buffered solution than in those receiving HCO3(-)-buffered solution. This suggests that the HCO3(-)-dependent mechanism of regulation may be depressed in diabetic rat hearts.

Amiloride↗
PubMed2003

[Effects of Na(+)-HCO3- symport on ischemia/reperfusion injury; Na+/H+ exchanger inhibitor and buffer composition].

We studied the impact of perfusate buffer composition on the relative degree of protection afforded by Na+/H+ exchanger (NHE) inhibition during ischemia as opposed to during reperfusion. Isolated rat hearts were perfused with bicarbonate- or HEPES-buffered medium. There was infusion of HOE 694 immediately before ischemia, during initial reperfusion, or during both of these periods. With bicarbonate-buffered medium, HOE 694 improved the post-ischemic recovery of left ventricular developed pressure (LVDP) when given before ischemia and before ischemia plus during reperfusion. In the presence of HEPES-buffered medium, however, HOE 694 significantly improved recovery of LVDP in all protocols. HOE 694 also provided an almost complete recovery of LVDP (88 +/- 9% vs 30 +/- 7% in controls) when given before ischemia plus during reperfusion. In conclusion, our results suggest that the influence of NHE activity during reperfusion on the extent of functional recovery is modulated significantly by perfusate buffer composition.

Animals↗
PubMed1997

[Surface photovoltaic spectroscopic investigation of Bacteriorhodopsin (BR)].

The surface photovoltaic spectra (SPS) of Bacteriorhodopsin-based films were systematically measured. The results show that the BR films are characterized by the specific photovoltaic effect. The SPS response of the BR films depends on the fabrication and humidity of the film. The intermediate M412 and O640 of BR are observed in the SPS of HEPES-containing BR film, indicating that HEPES has very important function to stabilize intermediate M412. The observations are explained in terms of the photocycle of BR and the mechanism of photovoltage generation.

Bacteriorhodopsins↗
PubMed1989

Use of the computer program EQUIL to estimate pH in model solutions and human urine.

The computer program EQUIL was designed to calculate relative supersaturations of solute components of common urinary stones. In an extended software version, quantitative consideration of charge balance for a priori or a posteriori pH estimation was added. The reliability of this computation was tested with hydrogen ion titration of buffer solutions containing HEPES [N-(2-hydroxyethyl)piperaizine-N'-ethanesulfonic acid] as well as samples of normal human urine. In the model solutions with HEPES, the difference between calculated pH values and the measured pH was smaller than 1.2% for any titration step within the buffer zone (pH 8.5-6.8). The pH values calculated for whole urine differed from the measured pH by 7% to 53%, and the calculated charge inbalance ranged from 2.6 to 9.6 mM. This net cation inbalance indicates that there is a need to account for other anionic components, including hippurate, amino acids, and isocitrate. In experimental solutions, charge balance calculations with EQUIL can be of great utility because they permit a priori estimation of pH or computation of the composition at a desired pH.

Buffers↗
PubMed1993

Quantitative analyses of the interaction between calcium ions and human serum albumin.

We examined the suitability of nine organic buffers for studying calcium binding to albumin by equilibrium dialysis. Results obtained with defatted human serum albumin showed that 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-([tris(hydroxymethyl)methyl] amino)ethanesulfonic acid were superior. Scatchard analysis of the experimental data from an extensive study performed in HEPES at pH 7.4 and 20 degrees C revealed (putting n(i) = 1, i = 1-4) k1 = 367 L/mol, k2 = 314 L/mol, k3 = 291 L/mol, and k4 = 179 L/mol. The very weak binding was characterized as n5 = 10 and k5 = 40 L/mol. The results were also analyzed in terms of stoichiometric association constants. The constants K1 and K2 were calculated to be 1513 and 647 L/mol, respectively, whereas the other constants were considered undeterminable. pH studies showed that in the interval 6.8-7.4, binding was not influenced by changes in acidity. Increasing pH to above the physiological value resulted in increased binding. At pH 8.0, k1 was increased almost fourfold, whereas k2 and k3 were approximately doubled. These findings indicate that the neutral to basic transition is important for the calcium-binding properties of albumin. The transition is a reversible, gradual conformational change of the protein at pH 6-9.

Buffers↗
PubMed1977

[Formation of spheriods by cultured human diploid cells].

When human lung diploid fibroblasts are grown in micro-wells in presence of the organic buffer HEPES in an atmosphere of 5% of CO2, the cells migrate and form spheroids. Histologic examination reveals the presence of fibroblastic and epithelioid cells in these spheroids. Signs of degeneration are seen in both cell types under the electron microscope. Following dissociation of the spheroids and their subculture in absence of HEPES, the cells reverse to their original fibroblast-like morphology and grow in monolayer.

Cell Aggregation↗