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PubMed2002

Non-catalytic role of carbonic anhydrase in rat intestinal absorption.

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.

Animals↗
PubMed2002

Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine.

The use of culture media of known composition are necessary for studying the role of trophic molecules. Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum. After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined. Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used. The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites. Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants. However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum. Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine. The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum.

Albumins↗
PubMed1998

Serotonin-stimulated calcium responses in human platelets: assay buffer dependency.

In Fura-2 labelled human platelets, the observed calcium response to serotonin stimulation is increased if the assay is conducted in a Krebs buffer compared with the HEPES buffer system usually used. The serotonin response in the Krebs buffer system was concentration dependent with an EC50 value of approximately 0.3 microM and was blocked by nanomolar concentrations of the serotonin2-receptor antagonist methiothepin. In a series of samples collected from 11 persons, a reasonable correlation was seen between the response to 1 microM serotonin and the serotonin2A-receptor density measured in corresponding membrane preparations. The calcium response to serotonin was reduced by treatment with hydrogen peroxide. It is concluded that the use of the Krebs buffer gives a more sensitive response of the Fura-2 loaded human platelets to serotonin stimulation than does the use of HEPES buffer and that the increased sensitivity is achieved without affecting the pharmacological properties of the response. For the response to thrombin, the response intensity is affected by the pH of the buffer used but is not sensitive to the buffer composition.

Blood Platelets↗
PubMed2001

Intracellular pH regulation in colonocytes of rat proximal colon.

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.

Ammonium Chloride↗
PubMed1986

Phosphate inhibition of the copper- and zinc-containing superoxide dismutase: a reexamination.

Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength.

Animals↗
PubMed1997

Structure and function of the xenobiotic substrate-binding site and location of a potential non-substrate-binding site in a class pi glutathione S-transferase.

Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.

Animals↗
PubMed1982

A collaborative study on the suitability of commercial, assayed plasmas for one-stage factor VIII assays.

A collaborative study was carried out to determine the suitability of commercial assayed plasmas for one-stage factor VIII assays. Activated partial thromboplastin times were performed on successive days on serially diluted paired freeze-dried reference plasmas (one sample buffered with HEPES), a freeze-dried test plasma and local laboratory standards-commercial assayed plasmas and in-house frozen pooled plasmas calibrated against commercial plasmas. The factor VIII activity levels of the study reference plasmas relative to average normal plasma were arrived at cumulatively by assaying them against a large number of individual fresh normal plasmas. Estimates of the activity of the test plasma relative to the claimed activity of local standards showed significant discrepancies from the expected value. However the estimates were more precise when the activity was assessed relative to the activity of the local standards derived by calibration against the reference plasmas. The study also showed that a HEPES buffered reference plasma may not contribute to inter-laboratory variability of one-stage factor VIII assays.

Adult↗
PubMed2001

Semen sample collection in medium enhances the implantation rate following ICSI in patients with severe oligoasthenoteratozoospermia.

Ejaculation in medium increases the proportion of antibody-free spermatozoa in semen samples containing anti-sperm antibodies and thereby enhances the fertilization rate in vitro. The aim of this study was to investigate whether this technique is also beneficial in semen samples with severe oligoasthenoteratozoospermia (OAT) where bacteria and detritus are often present. A prospective randomized controlled trial was carried out to study the results of sperm preparation and fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) for OAT. Of the 114 couples (one cycle per couple) studied between 1998 and 2000, 55 men were randomized to have semen collection into sterile dry pots (group A) and the remaining 59 had samples collected into 20 ml HEPES buffered Ham's F-10 medium with 10% human serum albumin (group B). In group B the ejaculates were incubated for 30 min and mixed gently. The samples were then processed by mild centrifugation and washing followed by a mini-swim-up technique. The ejaculates in group A were prepared by the swim-up procedure only. The overall fertilization rate was 71.8% and was similar in groups A (fertilization rate = 66.7%) and B (fertilization rate = 64.3%). In group A, 10/55 clinical pregnancies were recorded (pregnancy rate 18%), with an implantation rate (IR) of 6.9% per embryo. In group B, 16 of 59 patients conceived leading to significantly higher implantation (9.9%, P < 0.001) and clinical pregnancy rates (27%, P < 0.001). It is postulated that the addition of medium before liquefaction could inhibit the binding of bacteria and detritus to the sperm surface and may diminish DNA damage caused by reactive oxygen species, leading to improved efficiency of fertilization. The results demonstrate that the addition of HEPES buffered Ham's F-10 medium to sample collection pots significantly improves the pregnancy rate after ICSI in patients with severe OAT.

Buffers↗
PubMed1997

Drastic facilitation by alpha-latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i.

1. Latrotoxin (LTX, 1-3 nM) caused a gradual increase of the spontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs-Hepes solution containing 2.5 mM Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mM) and to acetylcholine (ACh, 30 microM). 2. The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs-Hepes deprived of CaCl2) induced secretory responses that gradually reached 400-800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mM K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100-200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]o or in electroporated cells. 3. Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole-cell Ca2+ currents or basal [Ca2+]i. In fact, LTX attenuated the K(+)- or ACh-evoked increases in 45Ca2+ uptake and [Ca2+]i. 4. It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+i transients, produced by the Na(+)-Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na(+)-dependent action potentials.

Acetylcholine↗
PubMed1994

Modulation of antagonist binding to histamine H1-receptors by sodium ions and by 2-amino-2-hydroxymethyl-propan-1,3-diol HCl.

1. NaCl (100 mM) reduced the potency of (+)-N-methyl-4-methyldiphenhydramine ((+)-QMDP) as an inhibitor of the binding of [3H]-mepyramine to histamine H1-receptors on guinea-pig cerebellar membranes to a greater extent than that of mepyramine, consistent with the greater inhibitory effect of Na+ on the binding of [3H]-QMDP than on the binding of [3H]-mepyramine. 2. The concentration of 2-amino-2-hydroxymethyl-propan-1,3-diol HCl (Tris, HCl) buffer, pH 7.5, present had little effect on the temelastine-insensitive binding of [3H]-mepyramine, but caused a concentration-dependent inhibition of the binding of [3H]-mepyramine sensitive to 1 microM temelastine (H1-receptor binding), with an approximate IC50 of 75 mM, assuming that complete inhibition would have been achieved. 3. Inhibition of [3H]-mepyramine binding by Na+ was more marked in 10 mM than in 50 mM Tris HCl and was not evident in 200 mM Tris HCl. 4. The Kd for the temelastine-sensitive binding of [3H]-mepyramine measured in 10 mM Tris HCl, 0.24 +/- 0.01 nM, was increased by 2.2 +/- 0.2 fold by 100 mM NaCl, without any significant change in the maximum binding (Bmax). The Bmax for [3H]-mepyramine was similarly unchanged in 50 mM Tris HCl, but the Kd was increased 2.5 +/- 0.2 fold. 5. The Kd for the temelastine-sensitive binding of [3H]-mepyramine was also increased in 50 mM,compared with 10 mM, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] KOH (HEPES.KOH)buffer (Kd 0.25 +/- 0.02 nm in 10 mM HEPES), but the evidence for an interaction between HEPES and Na+ was less clear.6. The effect of 100 mM NaCl on the inhibition of [3H]-mepyramine binding in 10 mM Tris HCl was examined for a range of antagonists. The decrease in potency caused by Na+ was greatest for triprolidine, (+)-chlorpheniramine and benzilylcholine (9.6-10.3 fold increase in K1 values) but the binding of mepyramine and promethazine was much less affected (1.8 and 1.9 fold increase in Kd respectively). The Kd for temelastine was not significantly changed. In contrast to the general decrease in antagonist affinity in the presence of Na+, the for MDL 16,455A (4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-alpha,alpha-dimethylbenzene acetic acid) was increased, but only by 1.5 fold.7. It is concluded that Na+ can act as an allosteric effector of the binding of antagonists at the histamine HI-receptor. Tris HCl also appears to have an allosteric action at the H1-receptor.

Animals↗
PubMed1984

The effect of two different buffers on the high affinity 3H-ethylketocyclazocine and 3H-SKF10047 binding to guinea pig brain membranes.

In vitro mu and delta opioid receptor binding is known to be influenced by ions. High affinity 3H-SKF10047 and 3H-ethylketocyclazocine binding sites are found in brain membranes and postulated to be similar to mu opioid receptor binding. To investigate this postulate, we have studied how the high affinity binding of 3H-SKF10047, 3H-ethylketocyclazocine, a tritiated mu agonist, mu antagonist and delta agonist is altered when the radioreceptor binding assay incubation buffer is changed. The binding of 3H-ethylketocyclazocine and the mu antagonist (3H-naloxone) is highest in isotonic HEPES buffer, while the binding of the mu (3H-dihydromorphine) and delta (3H-D-ala-D-leu-enkephalin) agonist is highest in hypotonic Tris-HCl buffer. 3H-SKF10047 binding is similar in the two buffers. The inhibition of 3H-ethylketocyclazocine, 3H-SKF10047 and tritiated mu and delta opioid ligands by seven unlabeled ligands is then compared in the two buffers. Morphine chloride is a more potent inhibitor of 3H-ethylketocyclazocine binding and tritiated mu ligand in hypotonic Tris-HCl buffer than in isotonic HEPES buffer. The potency of naloxone, nalorphine, SKF10047, D-ala-D-leu-enkephalin, cyclazocine and phencyclidine in inhibiting 3H-ethylketocyclazocine binding is independent of the buffer system. None of the seven unlabelled substances change potency with buffer change when inhibiting the 1.2 nM 3H-SKF10047 binding. In sum our results show that 1 nM 3H-ethylketocyclazocine binding is influenced by buffer change in a manner very similar to mu ligand binding, while the 1.2 nM 3H-SKF10047 binding is only slightly influenced by buffer change and therefore different from mu ligand binding.

Analgesics, Opioid↗
PubMed1990

Ionic control of intracellular pH in rat cerebellar Purkinje cells maintained in culture.

1. Intracellular pH (pHi) was measured in single rat cerebellar Purkinje cells maintained in primary culture using microspectrofluorescence analysis of the intracellularly trapped pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF). 2. The ratio of the fluorescence signals measured at 530 nm in response to an alternating excitation at 450 and 490 nm was calibrated using the K(+)-H+ ionophore nigericin. This calibration gave a steady-state pHi of 7.06 +/- 0.02 (S.E.M., n = 17) when cells were perfused by a 5% CO2-25 mM-HCO3(-)-buffered solution at an external pH of 7.40 at 37 degrees C. 3. Replacement of external chloride with gluconate in the presence of bicarbonate induced a cytoplasmic alkalinization of about 0.3 pH unit. This alkalinization was independent of external sodium and was greatly reduced by 0.5 mM-DIDS, indicating the presence of a chloride-bicarbonate exchange. 4. In bicarbonate-free (HEPES-buffered) solution the steady-state pHi was 7.37 +/- 0.02 (n = 19), significantly higher than in bicarbonate-buffered solution. Recovery from an intracellular acid load brought about by the ammonium chloride pre-pulse technique was blocked by the removal of external sodium or the addition of 1.5 mM-amiloride, indicating the presence of a sodium-hydrogen exchange. 5. In bicarbonate-buffered solution pHi recovery after an acid load was also completely blocked by addition of 1.5 mM-amiloride indicating the absence of a bicarbonate-dependent acid extrusion mechanism. 6. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) induced an amiloride-sensitive alkalinization of about 0.3 pH unit in bicarbonate-buffered solution but had no effect in HEPES-buffered solution. This observation suggests that in cultured Purkinje cells the sodium-hydrogen exchanger could be activated through a protein kinase C pathway only when pHi is maintained at a low physiological value by the activity of the chloride-bicarbonate exchange.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1975

Influence of oxygen and culture media on maintenance of whole hamster trachea organ cultures and replication of Sendai virus.

Effects of various oxygen concentrations and culture media on the maintenance of 4-day-old hamster trachea organ cultures and the yield of Sendai virus were studied. The basic media used were Eagle minimal essential medium, medium 199, and CMRL 1066 supplemented with glutamine and antibiotics and buffered with NaHCO3 or N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid (HEPES). In addition, each medium was evaluated under a gas phase of 5% CO2 and 95% O2, 5% CO2 and 45% O2 and 50% N2, or 5% CO2 and 95% air. Culturing of explants with CMRL 1066 and medium 199 buffered with HEPES in the presence of 5% CO2 and air proved most efficient; ciliary movement and ciliated surface epithelium were maintained for periods up to 27 days. No significant difference in the rate of replication of Sendai virus was seen in the three different media with the two buffer systems in the three different gaseous phases. The addition of 0.2% bovine serum albumin to the media yielded greater quantities of virus, up to 200-fold increase in titer without producing changes in ciliary function. A distinctive pattern of morphological changes was observed in explants of trachea epithelia inoculated with Sendai virus. These results suggest the practical application in the use of whole hamster trachea explants as a diagnostic aid in the isolation of Sendai virus from laboratory rodents.

Animals↗
PubMed1987

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey.

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ.

The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the modulation of SPQ fluorescence by commonly used buffers was investigated as a function of pH. Titration curves demonstrated SPQ quenching and yielded pK values characteristic of each buffer. from here, pH-independent Stern-Volmer constants, KQbase, were calculated for each of the morpholine derivatives, MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine derivatives, exhibited an additional pH-independent "molecular" quenching constant KmQ throughout the pH range 3-10. To study chloride fluxes, therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl, which is a function of both pH and buffer composition. Equations describing these relationships are presented. In conclusion, unless both pH and the buffer composition are taken into account, SPQ is unsuitable for studying the concomitant transmembrane fluxes of Cl- and H+.

Alkanesulfonates↗
PubMed1988

Acid-base effects on ileal sodium chloride absorption in vitro.

Previous work from this laboratory has shown that in the intact animal, ileal sodium chloride absorption is responsive to alterations in systemic acid-base balance. In vitro studies were undertaken to determine the nature of the active transport process affected. Paired, unstripped ileal tissues from Sprague-Dawley rats were mounted in modified Ussing chambers, and unidirectional 22Na and 36Cl fluxes were measured under short-circuited conditions (Isc). Acid-base effects were examined by changing either bathing solution partial pressure of CO2 (PCO2) or HCO3 concentration or by the addition of HCl or NaOH to a non-HCO3 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer. When pH was decreased from 7.6 to 7.1, net sodium absorption increased twofold regardless of the means by which pH was changed. This increase was primarily accounted for by an increase in mucosal-to-serosal flux. Net chloride absorption was affected in a similar way, but the increase in absorption was primarily accounted for by an increase in mucosal-to-serosal flux only in the HEPES buffer. These changes were accompanied by reductions in Isc and residual flux. When all groups were considered, mucosal-to-serosal Na (JNam----s) and net Na (JNanet) fluxes correlated strongly with bathing solution pH (r = 0.84 and 0.94, respectively, P less than 0.01). These data indicate that an active ileal sodium chloride absorptive process is specifically responsive to changes in bathing solution pH. In addition, pH may affect ileal electrogenic chloride, HCO3, and/or H secretion.

Absorption↗
PubMed1989

Intracellular pH regulation in IEC-6 cells, a cryptlike intestinal cell line.

Regulation of intracellular pH, pHi, was studied using microspectrofluorimetry of the pH-sensitive, fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in the rat intestinal crypt cell line, IEC-6. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions with pHi 7.25, treatment with a pulse of NH4Cl caused cells to acidify and then recover to control level. Because recovery was Na dependent, blocked by 1 mM amiloride, and unaffected by the presence and absence of Cl, it was likely because of a Na+-H+ exchanger. Cells were also acid loaded by changing from HEPES to HCO3-CO2-buffered solutions. pHi again recovered, but 1 mM amiloride reduced the rate of H+ efflux by only 47%. This HCO3-dependent, amiloride-insensitive H efflux required Na+ but not Cl- and was completely blocked by 200 microM [H2] 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We conclude that a Na+-HCO3- cotransporter was operative. Cl-free solutions caused pHi to increase from 7.19 to 7.41; this effect required the presence of exogenous HCO3-CO2 but not Na and was blocked by 200 microM [H2]DIDS. A Cl- -HCO3- exchanger is the most likely explanation for these data. All the pHi regulatory mechanisms are operative in NaCl-HCO3-CO2-buffered solutions. The Na+-H+ and Na+-HCO3- mechanisms are acid extruders, whereas the Cl- -HCO3- exchanger is an acid loader. These transporters may be important for generating HCO3 secretion by intestinal crypt cells.

Acids↗
PubMed1994

Role of diprotonated phosphate in evoking muscle reflex responses in cats and humans.

Lactic acid and H+ evoke muscle reflexes that raise sympathetic nerve activity. Whether these substances are direct afferent stimulants or markers for the acidification of other substances is unknown. Diprotonated phosphate (H2PO4-), a possible mediator of fatigue, increases as the cell acidifies and phosphate is produced. Its role in evoking muscle reflexes is unknown. We used 31P-nuclear magnetic resonance to measure forearm muscle H+ and H2PO4- and microneurography to measure muscle sympathetic nerve activity (MSNA, peroneal nerve) during a handgrip protocol designed to dissociate H+ from H2PO4-. Ischemic handgrip (50% maximal voluntary contraction x 2 min) was followed by a 1-min rest period during which the muscle was freely perfused. This was followed by a second bout of ischemic handgrip and a 5-min recovery. In seven of eight subjects, MSNA correlated with H2PO4-, whereas it correlated with pH in only one subject. To determine whether muscle reflex responses are evoked by H+, lactic acid, monoprotonated phosphate (HPO4(2-), or H2PO4-, we injected H+, lactate, H2PO4- [all 50 mM in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered to pH 6], and HPO4(2-) (50 mM, pH 7.5 in 10 mM HEPES) into the arterial supply of the triceps surae of the cat (n = 9) as we measured mean arterial blood pressure (MAP). H2PO4- increased MAP more than HPO4(2-), H+, or lactate (27.1 +/- 3.7 vs. 5.0 +/- 1.3, 4.6 +/- 3.1, and 7.7 +/- 3.2 rise in mmHg).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗