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PubMed1996

The actions of ouabain and lithium chloride on cytosolic Ca2+ in single chromaffin cells.

The effects of ouabain, Li+ and veratridine on the concentration of cytosolic free Ca2+ ([Ca2+]i) were studied in single fura-2-loaded bovine adrenal chromaffin cells. Superfusion of cells with ouabain (10 microM for 60 min) caused only a delayed mild increase of the Ca2+]i, from around 0.1 microM to 0.2-0.3 microM; this increase was Nao(+)-dependent. Replacement of all NaCl of the Krebs-Hepes solution by LiCl (144 mM) produced a gradual increase of [Ca2+]i, which remained elevated at a stable plateau of 0.4-0.5 microM for 40-50 min. When ouabain (in the presence of normal Nao+) or Li+ (in the absence of Nao+) was given in Krebs-Hepes solution containing no Ca2+, the reintroduction of 2.5 mM Ca2+ produced a fast elevation of the [Ca2+]i. In the case of ouabain-treated cells, the [Ca2+]i curve exhibited an initial phasic component which inactivated to a tonic component. omega-Conotoxin MVIIC (3 microM) and R56865 (10 microM) inhibited the phasic but not the tonic component. Veratridine (30 microM) induced large [Ca2+]i oscillations. Both ouabain or Li+ abolished such oscillations. These results are compatible with ouabain causing elevation of [Ca2+]i in bovine chromaffin cells through a dual mechanism, i.e. cell depolarisation and slowing down of the Na(+)-Ca2+ exchanger of their plasmalemma. Through its binding to the Na+ site on the Na(+)-Ca2+ exchanger, Li+ ions generate powerful Cai2+ signals that might be relevant to its known effects on neurosecretory mechanisms.

Adrenal Glands↗
PubMed1988

Na/H exchange in antral cells isolated from Necturus, rabbit, and guinea pig.

Mechanisms of intracellular pH (pHi) regulation were investigated using the pH-sensitive dye BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to measure pHi in isolated antral cells from Necturus, rabbit, and guinea pig. The BCECF fluorescence was calibrated in terms of pHi by using a nigericin-high potassium concentration method to equilibrate pHi and pHo. Average pHi for the cells of Necturus and rabbit was approximately 7.1 in a HEPES-buffered, HCO3-free Ringer's solution. Average pHi in Necturus cells in CO2/HCO3-buffered Ringer's solution was approximately 7.0. An ammonium-loading technique was used to acidify the cell to measure intrinsic buffering capacity (beta i) and to monitor pHi recovery. Average beta i = 28 mM per pH unit in Necturus and 42 mM per pH unit in rabbit cells in HEPES-buffered Ringer's solution. Recovery from an acid load was sodium-dependent, completely inhibited by 100 microM amiloride, and unaffected by changes in potassium concentration between 6 and 25 mM. The results provide evidence in support of the presence of an amiloride-sensitive Na/H exchanger in antral cells from rabbit, guinea pig, and Necturus.

Animals↗
PubMed1995

Secretin causes H+/HCO3- secretion from pig pancreatic ductules by vacuolar-type H(+)-adenosine triphosphatase.

BACKGROUND/AIMS: Secretin stimulates pancreatic ductules to secrete HCO3- into pancreatic juice and H+ into interstitial fluid. The aim of the present study was first to examine whether ductular H+ secretion is inhibited by micromolar concentrations of bafilomycin A1, which blocks vacuolar H(+)-adenosine triphosphatase by specific action, and secondly to test for evidence of ductular Na+/HCO3- cotransport. METHODS: Ductular H+ secretion was estimated from the rate of intracellular pH recovery after acid-loading (24 mmol/L NH4Cl) microdissected pancreatic ductules from pig, mounted in a flow-through perfusion chamber on the stage of a fluorescent microscope. Intracellular pH was measured using the fluorescent pH indicator 2'7'-bis (carboxyethyl)-5,6-carboxyfluorescein and dual-wave-length excitation of fluorescence. The ducts were superfused perfused with either HCO3(-)-free HEPES-containing buffers or HCO3(-)-containing buffers. RESULTS: Secretin (10(-8) mol/L) induced a net H+ secretion of 1.87 +/- 0.23 mumol.mL cell vol-1.min-1 that was blocked by 10(-6) mol/L bafilomycin A1 and was unaffected by Na+ substitution with choline using HEPES superfusion buffers. Secretin-stimulated ductules superfused with bicarbonate-containing, Cl(-)-free buffers showed Na(+)-dependent and 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid-inhibitable alkalinization of intracellular pH. CONCLUSIONS: Secretin causes H+/HCO3- secretion from pancreatic ductules by a mechanism involving vacuolar-type H(+)-adenosine phosphatase. Pancreatic ductules also show Na+/HCO3- cotransport, which may account for a small fraction of secreted bicarbonate.

Amiloride↗
PubMed1988

Effects of assay medium composition on macrophage-mediated tumor cell binding and lysis.

Activated macrophages express selective, high avidity tumor binding and cytotoxicity for tumor targets. The reported macrophage-mediated tumor binding and killing activities vary considerably among studies. Whereas, some of these studies utilized identical tumor target cells and similar sources of macrophages, the composition of the assay media used by different groups varied with respect to: type of medium (Eagle's minimal essential medium (EMEM), RPMI-1640, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEMLG), or with 4.5 g/l, glucose (DMEMHG)), concentration of serum (0-20%), and the addition of certain reagents (amphotericin B, pyruvate, and Hepes). The purpose of this study was to evaluate the effects of varying these parameters of medium composition on macrophage-mediated high (HA) and low avidity tumor binding and cytolysis. Tumor cytolysis was measured with an 18 h 51Cr release assay using peritoneal macrophages from C3H/HeN mice primed in vivo with pyran copolymer and further stimulated in vitro with lipopolysaccharide (10 ng/ml). P815 tumor cells were used as targets at a 10:1 effector:target ratio. Binding of targets to macrophages was determined by a method utilizing inverted centrifugation. Under optimal conditions, 2 x 10(5) macrophages bound as many as 17,600 +/- 3565 tumor cells and caused up to 50.4 +/- 3.6% cytolysis. Assays performed in DMEMHG compared with the other three media tested resulted in HA tumor binding and cytolysis which were decreased by up to 42.5% (P less than 0.05) and 64.3% (P less than 0.01), respectively, compared with the three other types of medium. The addition of pyruvate (1 mM) to EMEM with 5% fetal calf serum (FCS) stimulated 98.8% (P less than 0.01) and 50.6% (P less than 0.1) increases in tumor binding and cytolysis, respectively compared with EMEM/5% FCS alone, while Hepes (25 mM) stimulated 58.3% (P less than 0.01) and 37.5% (P less than 0.1) increases in these activities. Amphotericin B (2.5 micrograms/ml) completely abrogated tumor cytolysis, but it caused no change in tumor binding. Serum produced variable effects on macrophage-mediated tumor killing. Five of six lots of FCS inhibited tumor lysis, by 16 to 98% (32.6 +/- 28.6%; mean +/- SD). However, the same lot of FCS which inhibited cytolysis by 98% enhanced HA binding by 152% (P less than 0.05). Finally, several commercially available serumless medium preparations supported macrophage-mediated tumor binding; however, none of the serumless media tested supported macrophage-mediated tumor cytolysis. We conclude that common differences in assay medium composition can markedly alter macrophage-mediated tumor cell binding and cytolysis.

Amphotericin B↗
PubMed1986

Displaceable binding of [3H]l-glutamic acid to non-receptor materials.

[3H]L-glutamic acid binding to microfuge tubes and glass was investigated in four buffers. Background binding to these materials was negligible, but was increased by centrifugation or suction in Tris-HCl and Tris-citrate buffer. This binding was much less or eliminated when HEPES-KOH, or Tris-acetate buffer was used instead. [3H]L-glutamate binding to microfuge tubes was inhibited by L- but not D-isomers of glutamate and aspartate. DL-2-amino-7-phosphonoheptanoic acid also did not inhibit the binding. Other compounds which showed low to moderate inhibition were: N-methyl-D-aspartate, quisqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, and 2-amino-4-phosphonobutyrate. Binding was inhibited by denatured rat brain membranes. A protein-dependent [3H]glutamate binding was obtained with a repeatedly frozen-thawed membrane preparation when binding was done in Tris-acetate buffer. It is recommended that Tris-acetate or HEPES-KOH buffer should be used in the glutamate binding assay. If Tris-HCl or Tris-citrate buffer is used, appropriate control experiment should be done to correct for binding to microfuge tubes or glass fiber filters.

Animals↗
PubMed1988

Covalent immobilization of the estrogen receptor to a cationic N-hydroxysuccinimide ester derivative of agarose.

Covalent immobilization of the soluble estrogen receptor from a rabbit uterus to N-hydroxysuccinimide ester derivative of agarose is shown. At first, the condition for the immobilization reaction was examined. The non-immobilized receptor was extracted with 0.4 M NaCl-containing medium. Sixty seven to 80% of the input receptor were immobilized within 30 min at 0 degrees C in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4). The immobilized [3H]estradiol(3,17 beta-dihydroxy-1,3,5(10)-estratriene)-receptor complex was stable for at least 24 h. The optimum pH for immobilization was 7.4. Ca2+ or Na+ ions in the reaction media decreased the yields in immobilization of the receptor to the reagent with an electrostatically positive spacer arm. Next, influences of immobilization on the receptor were examined. The dissociation rate of [3H]estradiol from the immobilized receptor was a little slower than that from the native receptor. The estrogen-free immobilized receptor was saturated by incubating with 10 nM [3H]estradiol for 10 h at 0 degrees C in 0.1 M HEPES (pH 7.4). From Scatchard plot analysis, it was found that the hormone binding affinity in the immobilized receptor decreased to approximately one-fourth of that in the native receptor.

Animals↗
PubMed1993

pH regulation in frog cones studied by mass receptor photoresponses from the isolated retina.

Mass cone photoresponses were recorded across the aspartate-treated frog retina under treatments chosen to affect putative pH-regulating mechanisms. The saturated response amplitude (Umax) was found to be a monotonically increasing function of perfusion pH in the range 7-8, and thus presumably of intracellular pH (pHi). Accepting that Umax can be used as an index of pHi changes, two results indicate the importance of bicarbonate transport for preventing intracellular acidification: (1) bicarbonate-buffered (6 mM HCO3- + 6 mM HEPES) perfusate increased Umax compared with nominally bicarbonate-free perfusate (12 mM HEPES); (2) the anion transport blocker DIDS (0.1 mM) caused a strong decrease in the amplitude of photoresponses. Substitution of 95 mM chloride by gluconate in the perfusing fluid boosted photoresponses indicating that at least part of the bicarbonate transport involves HCO3-/Cl- exchange. Amiloride (2 mM) also caused a decrease of photoresponse amplitude, which suggests that Na+/H+ exchange contributes to pHi regulation. In all these respects, cones behaved similarly to rods. Cones differed from rods (in the intact retina) in that addition of 0.5 mM of the carbonic anhydrase inhibitor acetazolamide reduced (never augmented) photoresponses. The difference is considered in relation to the presence of carbonic anhydrase in cone, as opposed to rod, outer segments.

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

Comparison of the in vitro effect of eicosapentaenoic acid (EPA)-derived lipoxygenase metabolites on human platelet function with those of arachidonic acid.

Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1986

Eicosapentaenoic acid metabolism in human and rabbit anterior uvea.

Eicosapentaenoic acid (EPA) metabolism into 3 series cyclooxygenase and 5 series lipoxygenase products was assessed in human and rabbit anterior uvea. Both tissues synthesized 3 series cyclooxygenase products such as delta17 6-keto-PGF1 (PGI3 metabolite), PGE3 alpha, PGE3, PGD3 and TxB3 (a stable product of TxA3) and lipoxygenase products 12-hydroxyeicosapentaenoic acid (HEPE), 5-HEPE and 5,12-diHEPE from 14C-EPA. EPA-derived cyclooxygenase product synthesis was considerably greater than the formation of lipoxygenase products from EPA in both tissues.

Alprostadil↗
PubMed1990

Site selectivity in the binding of inorganic anions to serum transferrin.

Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding.

Anions↗
PubMed1992

The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

(1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

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

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature.

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.

Animals↗
PubMed1983

Basal and dopamine-inhibited prolactin secretion by rat anterior pituitary cells: effects of culture conditions.

Culture conditions for rat pituitary cells were investigated which would result in high PRL synthesis and secretion with maintenance of dopamine-mediated inhibition of PRL secretion. From five commercially available media, RPMI resulted in the highest PRL content and secretion, but no inhibition of PRL secretion by dopamine was observed. MEM with Earle's salts fulfilled best our requirements for culturing functional PRL-secreting cells. PRL secretion was not affected by variations in the concentration of fetal calf serum, but was positively correlated with increasing horse serum concentrations. TRH-induced PRL release increased with increasing serum concentrations and was positively correlated with the concentration of 17 beta-estradiol in the culture medium (P less than 0.0025). An increase in the sodium bicarbonate concentration from 0.85 to 3.0 g/l resulted in a 4-fold stimulation of PRL synthesis and in a 27-fold stimulation of PRL secretion. However, at bicarbonate concentrations above 2.6 g/l, inhibition of PRL secretion by 500 nM dopamine was lost. The addition of 20 mM Hepes to the culture medium decreased basal PRL secretion by 48 +/- 13% (P less than 0.01), while dopamine inhibition of PRL secretion was reduced from 49 +/- 10% to 24 +/- 8% (P less than 0.05). When an increasing number of pituitary cells was cultured in a constant volume, PRL secretion expressed per cell increased up to 0.3-0.4 X 10(6) cells/dish/2 ml. With higher cell concentrations of up to 1 X 10(6) cells/dish, PRL secretion per cell diminished significantly, which indicates a direct negative feedback of high medium PRL on the PRL-secreting pituitary cells. In this culture system dopamine inhibited PRL secretion over a 4 h period in a dose-dependent manner (IC50 20 nM), while no paradoxical stimulation of PRL secretion was observed with low dopamine concentrations. However, a 25% stimulation (P less than 0.05) of PRL secretion by 0.1 nM dopamine could be obtained by addition of 0.01% ascorbic acid, which by itself decreased basal PRL secretion by 49% (P less than 0.01). Thus, tissue culture conditions that result in high PRL production are not necessarily the best choice, since dopamine-mediated inhibition of PRL secretion is another important parameter for the functioning of lactotrophs in culture. The best compromise is MEM with 2.2 g/l of sodium bicarbonate, without Hepes buffer and supplemented with 10% FCS.

Animals↗
PubMed1982

Interactions of cellular glycosaminoglycans with plasma fibronectin and collagen.

The interactions of metabolically radiolabelled glycosaminoglycans, isolated from Swiss mouse 3T3 and SV3T3 cells, with plasma fibronectin and collagen were studied by affinity column chromatograph in Hepes-buffered saline (150 mM NaCl in 10 mM Hepes buffer). Cell surface heparan sulfate glycosaminoglycan bound to plasma fibronectin and to native calf skin collagen in the absence of Ca2+ and Mg2+ with maximal binding at slightly acidic pH. Elution should be effected with elevated ionic strength. 3T3 and SV3T3 heparan sulfate coeluted when exposed to a salt concentration gradient. The core protein of the heparan sulfate proteoglycan did not bind to either fibronectin or collagen. Reference standard chondroitin 4-sulfate and chondroitin 6-sulfate had no effect upon heparan sulfate binding, whereas 90% of the heparan sulfate bound to fibronectin or collagen was eluted with heparin and 60% removed from fibronectin with dermatan sulfate. Cell surface chondroitin sulfate proteoglycan bound only to collagen at acidic pH and no interaction was seen with fibronectin. Trypsin treatment of chondroitin sulfate proteoglycan reduced the binding to collagen. Cell surface hyaluronic acid did not display any affinity for either fibronectin or collagen.

Animals↗
PubMed1992

Factors affecting the manganese and iron activation of the phosphoenolpyruvate carboxykinase isozymes from rabbit.

Timed assays in which GTP and GDP were separated and quantitated by HPLC were developed and used to study the metal activation of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase purified from rabbit liver. These assays allowed both directions of catalysis to be studied under similar conditions and in the absence of coupling enzymes. The mitochondrial enzyme is rapidly inactivated by preincubation with Fe2+, as had been shown previously for the cytosolic isozyme. The greatest activation by Fe2+ was obtained by adding micromolar Fe2+ immediately after enzyme to form the complete assay mixture that also contained millimolar Mg2+. In the direction of synthesis of OAA from Pep, the K0.5 values for Mn2+ and Fe2+ were in the 3-7 microM range when a nonchelating buffer, Hepes, was used. The buffer used strongly affected activation by Fe2+ at pH 7.4; activation was eliminated in the case of phosphate and K0.5 increased several-fold over that obtained with Hepes when imidazole was used. In non-chelating buffer, the pH optimum was near 7.4 for both isozymes and for both directions of catalysis. However, the near optimal pH range extended below 7.4 for the direction of oxaloacetate synthesis while the range was above 7.4 for Pep synthesis. In the direction of oxaloacetate synthesis: (1) Both isozymes required the presence of micromolar Mn2+ or Fe2+ in addition to millimolar Mg2+ in order to shown significant activity. (2) Fe2+ was as effective an activator as Mn2+ at pH 7 and below. In the direction of Pep synthesis: (1) Micromolar Mn2+ was a much better activator than Fe2+ at the higher pH values needed for optimal activity in this direction. (2) With increasing pH, decreasing activation was obtained with Fe2+ while the activity supported by Mg2+ alone increased. The results demonstrate the potential for regulation of either isozyme of Pep carboxykinase by the availability of iron or manganese.

Animals↗
PubMed1988

Stimulus-induced extracellular pH transients in the in vitro turtle cerebellum.

In a number of CNS preparations, neuronal activation has been shown to result in a rapid extracellular alkaline transient, followed by a prolonged acid shift. The isolated turtle cerebellum was used to investigate the early alkaline transient. Double-barreled ion-sensitive microelectrodes for H+, K+ and tetramethylammonium were used to measure field potentials and extracellular ion and volume shifts in response to bipolar electrical stimulation of the parallel fibers. Transition from 15 mM HEPES to 35 mM HCO3- -buffered Ringer decreased the amplitude of the alkaline shift, presumably due to a marked increase in extracellular buffering power. In HEPES-buffered Ringer, repetitive stimulation produced alkaline shifts as large as 0.3-0.4 pH. Single shocks produced an alkaline shift of 0.006 +/- 0.0002 pH with a latency as short as 70 ms. Kynurenic acid (an excitatory amino acid antagonist), or Mn2+, blocked the alkaline shift and the postsynaptic component of the field potential. The alkaline shift was not blocked by the Na-H exchange inhibitor amiloride. The relationship between pHo and extracellular volume transients was studied using tetramethylammonium as an extracellular volume indicator. In nominally HCO3-free Ringer, stimulation at 5 Hz for 10 s caused a decrease in extracellular volume of 3.0 +/- 0.2 per cent. The volume transient was unaffected by 3 mM Mn2+, while the alkaline shift was completely abolished. The data for the alkaline shift are consistent with a channel-mediated transmembrane flux of proton equivalents. The size of the pH change and the underlying perturbation it represents, indicate that acid-base shifts may be a functionally important consequence of neuronal activity.

Action Potentials↗
PubMed1990

Measurement of protease thermostability, twitching motility and colony size of Bacteroides nodosus.

Total extracellular protease activity of Bacteroides nodosus in TAS liquid culture varied directly with cell mass and buffer concentration between 20 and 50 mM HEPES, MOPS and TES buffers, but not with Tris which gave anomalous high cell counts, nor with Na2Co3 which showed a decline of protease activity and cell mass. The stability of HEPES-buffered crude protease preparations were estimated on the basis of temperature or Ca2+ activity. Variation of the estimates for cellular twitching was greater than that for colony diameter in benign and virulent strains of B. nodosus. Surface translocation, quantified on the basis of colony diameter, reached a limit after 72 h incubation on modified TAS agar, ranging from 0.04 to 0.14 mm per h for six isolates tested.

Animals↗
PubMed1995

Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes.

L-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their Fab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in KD with increasing temperature and a positive entropy value between 20 and 35 degrees C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.

Adsorption↗