Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HEPES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed2000

Discovery of an 11 (R)- and 12(S)-lipoxygenase activity in ovaries of the mussel Mytilus edulis.

Eicosanoid biosynthesis was investigated in mussel gonads by incubation of tissue homogenates with radiolabeled arachidonic acid and analysis of the products by radio-high-performance liquid chromatography. No radiolabeled metabolites were formed in homogenates of testes, but two major metabolites were synthesized by ovarian preparations. The radiolabeled metabolites were analyzed by mass spectrometry and chiral chromatography and identified as 11 (R)-hydroxy-5,8,1 2,14-eicosatetraenoic acid and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid. In addition, four other nonlabeled metabolites, formed from endogenous substrates, were detected in ovarian extracts. Their structures, determined by mass spectrometric analysis, were the corresponding 11- and 12-hydroxy analogs formed from eicosapentaenoic acid (11-HEPE and 12-HEPE) and 9-hydroxy-6,10,12,15-octadecatetraenoic acid (9-HOTE) and 13-hydroxy-6,9,11,15-octadecatetraenoic acid formecl from stearidonic acid. The biosynthesis of the 11 - and 12-hydroxy products was calcium dependent, localized to the 100,000 x g supernatant cell fraction, and was inhibited by nordihydroguaiaretic acid, but not inhibited by the prostaglandin synthase inhibitors aspirin and indomethacin, or the monoxygenase inhibitor proadifen. Together these data suggested that both the 11 (R)- and 12(S)-hydroxy products were formed from lipoxygenase-type enzymes. Incubation of homogenates of immature ovaries with eicosapentaenoic acid revealed the major product to be I2-HEPE, whereas in mature ovaries mainly 11-HEPE was formed. Extraction of spawned eggs with methanol revealed that predominantly 11-HEPE and 9-HOTE were formed from endogenous substrates. This study shows that female gonads of the mussel express an 11(R)- and 12(S)-lipoxygenase activity whose expression is dependent on differentiation of the ovary.

Animals↗
PubMed1988

A 119Sn Mössbauer spectroscopic study on the interaction of dimethyltin (IV) derivatives with rat hemoglobin, and of related model systems in aqueous solution.

In the context of a study of the molecular basis of the antileukemia (murine) activity of diorganotin (IV) compounds, the interaction with rat hemoglobin (selected as a model protein) of the representative terms dimethyltin dichloride, dimethyltin glycylglycinate (Me2SnGlyGly), and dimethyltin L-cysteinate (Me2Sn-Cys) has been investigated by 119Sn Mössbauer spectroscopy. In order to possibly determine the reaction pathway, aqueous model systems in Hepes buffer at pH 7.4 were also considered. The structural characteristics of reactants and products were advanced on the basis of semiempirical calculations of Mössbauer nuclear quadrupole splitting parameters, delta E, by the point-charge model approach. In aqueous Hepes at pH 7.4, evidence was obtained for the formation of the five-coordinated species, trigonal bipyramidal type (tbp), Me2Sn(OH)2.Hepes(II), Me2Sn(OH)(GlyGly).Hepes(III), and Me2Sn(OH)Cys(IV) (see Fig. 1). Equatorial groups or atoms would be the Me radicals, as well as OH, N(peptide), and S(thiol), respectively. Hepes would coordinate to tin in axial position through the tertiary amino nitrogen, while cysteine would behave as a bidentate chelating agent, with an axially located amino group. Species (II), (III), and (IV) react with cysteine in aqueous Hepes at pH 7.4, yielding Me2Sn(OH)Cys(IV), as well as Me2SnCys2(V), where tin would be embedded into a tbp structure due to one cysteine probably chelating (equatorial S thiol and axial amino nitrogen), and one monodentate through S thiol. Species (II), (III), and (IV) react analogously with rat hemoglobin, primarily through the S thiol of a cysteine side chain, yielding pellets where the environment of tin could be tetrahedral, such as in Me2Sn(OH)(S thiol), (VI), and tetrahedral (IX) or tbp (V) in Me2Sn(Cys)(S thiol), where Cys would act either as chelating or monodentate. Further reaction of (VI) and (IX) could involve imidazole nitrogen atoms, N het, of histidine side chains, forming tetrahedral Me2Sn(S thiol)(N het), (VIII), or tbp Me2Sn(OH)(S thiol)(N het), (VII), and Me2Sn(Cys)(S thiol)(N het), (V) (see Figs. 1 and 5).

Animals↗
PubMedDate not supplied

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride.

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.

Animals↗
PubMed1991

pH regulation after acid load in primary cultures of mouse astrocytes.

Intracellular pH (pHi) recovery in primary cultures of mouse astrocytes after acid-loading was studied with the ion transport inhibitors (amiloride, SITS, acetazolamide, ouabain and bumetanide), and by reducing the concentration of Na+ or Cl- in HCO3- -free HEPES-buffered (HEPES) and in HCO3-/CO2 Hanks' balanced salt solution (HBSS). The pHi of astrocytes exposed to 15 mM NH4Cl decreased abruptly and began to recover slowly after 5 min. Exposure of the cells to NH4Cl for 2 min and reincubation in HEPES HBSS decreased pHi further within 1-2 min after removal of NH4Cl; pHi then recovered toward the control value. Cultures exposed to HCO3-/CO2 HBSS (10 mM/2%) showed changes in pHi in the opposite direction. These responses are unique to astrocytes and differ from those occurring in most other cells. Recovery of pHi after NH4Cl prepulse was markedly inhibited in low-Na+ and in amiloride-containing HEPES HBSS. Ouabain also reduced pHi recovery rate; however, SITS, acetazolamide and bumetanide did not. Therefore, Na(+)-H+ exchange is the major process for pHi recovery from acidification in HCO3- -free solution. In HCO3-/CO2 HBSS pHi recovery was markedly inhibited by SITS and acetazolamide, but not by amiloride, ouabain, or bumetanide. The inhibitory effect of SITS on pHi recovery was enhanced in low-Na+ HBSS. These results indicate that both Na+ and HCO3- are directly related to pHi recovery in HCO3-/CO2 solution after acid-load. Low-Cl HEPES HBSS and low-Cl HCO3-/CO2 HBSS media did not alter pH recovery rate. Thus, pHi recovery after acid-load is not Cl- -dependent, and therefore, does not involve a Na(+)-dependent Cl- -HCO3- exchange process. It appears that mouse astrocytes possess 3 acid-regulating systems: Na(+)-H+ exchange, Na(+)-HCO3- co-transport and Na(+)-independent Cl- -HCO3- exchange.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1986

Cardiac cell action potential duration is dependent upon induced changes in free Ca2+ activity during pH changes in vitro.

We examined how changes in solution pH alter myocardial cell action potentials (AP) with and without changes in free [Ca2+] caused by pH induced effects on calcium binding. Guinea pig ventricular tissue was isolated, superfused either with Krebs-Ringer (K-R) bicarbonate, phosphate buffered solution, or with Hepes buffered solution, and electrically paced during control (5% CO2 in O2), acidic (12% CO2), and alkalotic (0% CO2) conditions. Action potentials were recorded with intracellular microelectrodes. Extracellular free [Ca2+] was measured with a calcium ion selective electrode and total soluble calcium was measured by ultrafiltration and spectrophotometry. With a total [CaCl2] of 2.5 mM in the K-R solution, we found a free [Ca2+] of 2.14 mM at pH 7.44 (control), 2.48 mM at pH 6.97 and 1.60 mM at pH 8.19; total soluble calcium concentration was 2.00 mM at pH 8.19. In the Hepes solution, free [Ca2+] was only slightly altered (2.42 to 2.55 mM) within this pH range. Equivalent acidosis of either K-R or Hepes suffusate significantly, and similarly, prolonged the AP and its refractory period. Alkalosis of the Hepes suffusate shortened the AP; but equivalent alkalosis of the K-R suffusate prolonged the AP as did a reduction of [CaCl2] in Hepes suffusate from 3.0 to 1.5 mM at pH 7.43. Our study demonstrates that a paradoxical increase in APD occurs because free Ca2+ ion activity falls in K-R solution and overrides the effect of alkalosis alone to decrease APD.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1998

Separation of motile spermatozoa from frozen-thawed buffalo semen: swim-up vs filtration procedures.

Three experiments were conducted to maximize the recovery rate of motile spermatozoa from frozen-thawed buffalo semen. In Experiment 1, the swim-up of motile spermatozoa was performed in the presence or absence of HEPES in TALP medium and CO2 in the environment. The recovery rate of motile spermatozoa in TALP medium (control), TALP + HEPES + CO2, TALP + HEPES and TALP + CO2 was 15, 18, 12 and 10%, respectively (P > 0.05), with sperm motility at 87, 89, 90 and 90%, respectively (P > 0.05). In Experiment 2, the pH of TALP medium was adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and swim-up procedure was performed in the presence of HEPES and CO2. The recovery rate of motile spermatozoa at different pH was 14, 20, 24, 27 and 16%, respectively (P < 0.05). Motility of separated spermatozoa was 88, 91, 90, 89 and 90%, respectively (P > 0.05). In Experiment 3, the efficiency of ion-exchange filtration and Swim-up procedure in separating motile spermatozoa from frozen-thawed buffalo semen was compared. The recovery rate of motile spermatozoa was 95% in filtration procedure and 33% in swim-up procedure (P < 0.005). In all experiments, normal acrosomes did not vary due to treatments (P > 0.05). In conclusion, HEPES and CO2 had no significant effect on swim-up of buffalo spermatozoa. The pH 8.5 of TALP improved the recovery rate of motile spermatozoa in swim-up procedure. The ion-exchange filtration was found superior to swim-up procedure in harvesting maximum number of motile spermatozoa from frozen-thawed buffalo semen (95 vs 33%; P < 0.001).

Animals↗
PubMed1995

Reduction of chromium(VI) by ascorbate leads to chromium-DNA binding and DNA strand breaks in vitro.

Chromium(VI) is a known human carcinogen which requires intracellular reduction for activation. Ascorbate (vitamin C) has been reported to function as a major reductant of Cr(VI) in animals and cell culture systems. The reaction of Cr(VI) with varying concentrations of ascorbate was studied under physiological conditions in vitro in order to determine the types of reactive intermediates produced and to evaluate the reactivity of these intermediates with DNA. Reactions of 1.8 mM Cr(VI) with 0-18 mM ascorbate at pH 7.0 in N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES; 0.10 M) and tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl; 0.050 M) buffers were studied by electron paramagnetic resonance and UV/visible spectroscopy. Cr(V) and carbon-based free radical adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were observed at 0.5 to 1 and 1 to 1 reactions of ascorbate to Cr(VI). Levels of Cr(V) were higher for reactions in HEPES buffer, and levels of carbon-based radicals were higher in Tris.HCl buffer. Levels of Cr(IV) and Cr(III) increased with increasing concentration of ascorbate in both buffers. Reaction of Cr(VI) with varying ascorbate in the presence of calf thymus DNA or pBR322 DNA resulted in Cr-DNA adducts and plasmid relaxation, respectively. Maximum binding of Cr to DNA was observed for the 1:1 reaction ratio of Cr(VI) with ascorbate in both HEPES and Tris.HCl buffers, but total Cr bound to DNA was 8-fold lower in Tris.HCl than HEPES buffer. Preincubation of Cr(VI) with ascorbate before reaction with DNA decreased Cr-DNA binding to background levels. Preincubation of Cr(III) with ascorbate resulted in only low Cr-DNA binding. Levels of Cr-DNA binding were higher with single-stranded vs double-stranded DNA. Reactions with 14C-labeled ascorbate produced no cross-linking of ascorbate to DNA. Maximum plasmid relaxation was observed for the 1:1 ascorbate to Cr(VI) ratio in both buffers; however, single-strand breaks were 2-fold higher in Tris.HCl than HEPES buffer. Reactions with plasmid in the presence of DMPO quenched formation of single-strand breaks. Interpretation of these results in light of the spectroscopic studies suggested that Cr(V) and carbon-based radicals were responsible for Cr-DNA adducts and DNA single-strand breaks, respectively.

Ascorbic Acid↗
PubMed2000

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Acetaminophen↗
PubMed1993

Regulation of intracellular pH in the perfused heart by external HCO3- and Na(+)-H+ exchange.

Both Na(+)-dependent HCO3- influx and the Na(+)-H+ antiport have been shown to contribute to recovery from intracellular acidosis in avian and mammalian cardiac tissue. We have investigated the participation of these mechanisms in the recovery of intracellular pH (pHi) after an acid load in the Langendorff-perfused ferret heart. pHi was measured from the phosphorus-31 nuclear magnetic resonance chemical shift of 2-deoxy-D-glucose 6-phosphate. Basal pHi was higher in HCO(3-)-buffered solution (7.05 +/- 0.01; n = 8) than in nominally HCO(3-)-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution (6.98 +/- 0.02; n = 9). Addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a significant fall in pHi in HEPES solution (6.91 +/- 0.02; n = 5) but not in HCO3- solution (7.02 +/- 0.02; n = 5). Intrinsic intracellular buffering capacity in 0 Na(+)-HEPES solution was 37 +/- 2 mmol/l (n = 4), and additional buffering due to HCO(3-)-CO2 was approximately 13 mmol/l in HCO3- solution. After an intracellular acidosis induced by an NH4Cl prepulse, the proton efflux rate (JH) at pHi 6.90 was 0.5 +/- 0.2 nmol.l-1.min-1 (n = 14) in HEPES solution and 1.2 +/- 0.4 mmol.l-1.min-1 (n = 13) in HCO3- solution. The addition of 1 microM EIPA effectively blocked proton efflux in HEPES solution (JH < 0.1 mmol.l-1.min-1; n = 8), whereas it slowed pHi recovery in HCO3- solution (JH = 0.6 +/- 0.2 mmol.l-1.min-1; n = 9). There was no recovery of pHi in Na(+)-free HCO3- solution (JH < 0.1 mmol.l-1.min-1; n = 3). The Na(+)-H+ antiport and a mechanism requiring both external Na+ and HCO3- each contribute approximately 50% to proton efflux at pHi 6.90 during the recovery from intracellular acidosis in the isolated perfused mammalian heart.

Acids↗
PubMed1997

Ischemic preconditioning and intracellular pH: a 31P NMR study in the isolated rat heart.

The aim of these studies was to investigate whether manipulation of intracellular pH affects preconditioning in isolated buffer-perfused rat hearts. Control and preconditioned [PC; 3 min of ischemia (I) + 3 min of reperfusion (R) + 5 min of I + 5 min of R or 4 x (5 min of I + 5 min of R)] hearts were subjected to two different protocols expected to alter intracellular pH during the sustained ischemic insult: 1) increased extracellular buffering capacity with the addition of 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) to the buffer to alleviate acidosis and 2) increased preischemic glycogen content to exacerbate acidosis. All hearts were subjected to 40 min of I + 40 min of R. 31P nuclear magnetic resonance was used to measure ATP, phosphocreatine, Pi, and intracellular pH. Despite a significantly better recovery of function in all PC groups, there were no significant differences in intracellular pH (rate-pressure product = 60 +/- 5, 66 +/- 10, 42 +/- 5, and 57 +/- 8% of baseline in PC, 4 x 5 PC, PC + HEPES, and PC fasted hearts, respectively, compared with 36 +/- 9, 17 +/- 7, and 20 +/- 10% of baseline in control, control + HEPES, and control fasted hearts, respectively; pH at end ischemia: control, 6.31 +/- 0.02; PC, 6.35 +/- 0.03; 4 x 5 PC, 6.35 +/- 0.04; control + HEPES, 6.40 +/- 0.10; PC + HEPES, 6.56 +/- 0.07: control fasted, 6.46 +/- 0.03; PC fasted, 6.43 +/- 0.01). No significant differences were observed among groups in ATP, phosphocreatine, or Pi on reperfusion. Thus the mechanism of preconditioning in glucose-perfused hearts does not depend on an alleviation of intracellular acidosis during the sustained ischemic period. Furthermore, under the conditions of this study, intracellular pH during ischemia did not predict functional recovery on reperfusion.

Animals↗
PubMed2006

Effect of pH on trout blood vessels and gill vascular resistance.

pH is recognized as a modulator of vascular smooth muscle (VSM) tone in mammalian vessels, but little is known about its effects on fish VSM. We investigated the effects of extracellular and intracellular pH (pH(o) and pH(i), respectively) on isolated vessels from steelhead and rainbow trout (Oncorhynchus mykiss, Skamania and Kamloops strains, respectively) and of pH(o) on perfused gills from rainbow trout. In otherwise unstimulated (resting) efferent branchial (EBA) and coeliaco-mesenteric arteries (CMA), anterior cardinal veins (ACV) and perfused gills, increasing pH(o) from 6.8 to 8.8-9.0 produced a dose-dependent contraction or increase in gill resistance (R(GILL)) with an estimated half-maximal response of 8.0-8.2. pH(o) interactions with other contractile stimuli were agonist specific; more force was developed at low pH(o) in ligand-mediated (arginine vasotocin) contractions, whereas depolarization-mediated (40-80 mmol l(-1) KCl) contractions were greatest at high pH(o). Increasing pH(i) by application of 40 mmol l(-1) NH4Cl produced sustained contraction in afferent branchial arteries (ABA) suggesting that these vessels could not readily restore pH(i). NH(4)Cl application only transiently contracted EBA and CMA in Hepes buffer, whereas it produced a slight, but prolonged, relaxation of EBA and CMA in Cortland buffer. The buffer effect was due to the presence of Hepes and in this environment EBA and CMA appeared to readily restore pH(i). Increasing pH(i) in KCl-contracted EBA in Hepes produced an additional contraction, whereas ligand-contracted (thromboxane A2 analog, U-46619) EBA relaxed. Reducing pH(i) (NH(4)Cl washout) transiently contracted resting EBA and CMA in both Hepes and Cortland buffer. NH4Cl washout produced an additional, transient contraction of both KCl- and U-46619-contracted EBA in Hepes. EBA contractions produced by increased pH(i) depend primarily on intracellular Ca2+, whereas both intracellular and extracellular Ca2+ contributed to the response to decreased pH(i). Three cycles of perfusate acidification (pH(o) 7.8 to 6.2 and back to 7.8) reproducibly halved, then restored R(GILL) with no adverse effects, indicating that this was not a pathophysiological response. These studies show that the general effects of pH on VSM are phylogenetically conserved from fish to mammals but even within a species there are vessel-specific differences. Furthermore, as fish are exposed to substantial fluctuations in environmental (and therefore plasma) pH, the obligatory response of fish VSM to these changes may have substantial impact on cardiovascular homeostasis.

Ammonium Chloride↗
PubMed2000

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Animals↗
PubMed1990

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3- transporters by Ca2(+)-mobilizing agonists.

Pancreatic acini loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca2(+)-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, a while in HCO3(-)-buffered medium pHi is determined by the combined activities of a Na+/H+ exchanger, a Na(+)-HCO3- cotransporter and a Cl-/HCO3- exchanger. In this study we found that stimulation of acini with Ca2(+)-mobilizing agonists in HEPES or HCO3(-)-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPES-buffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H+ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCO3(-)-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO3(-)-buffered medium: (a) recovery from acid load in the presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) (Na+/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO3- influx and efflux, respectively, by Na(+)-HCO3- cotransport) were inhibited; and (c) HCO3- influx and efflux due to Cl-/HCO3- exchange, which was measured by changing the Cl- or HCO3- gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO3- exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO3- influx due to Na(+)-HCO3- cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO3(-)-buffered medium, as much as treating the acini with H2DIDS resulted in similar agonist-mediated pHi changes in HEPES- and HCO3(-)-buffered media. The effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Bicarbonate-dependent amantadine transport by rat renal cortical proximal and distal tubules.

Transport of the achiral cationic drug amantadine by purified renal cortical rat tubules was investigated in bicarbonate [Krebs-Henseleit solution (KHS)] and nonbicarbonate buffers. Values of the apparent Km for amantadine accumulation were higher (P < .001, mean +/- S.E., n = 5) for all nonbicarbonate buffers compared with those in KHS in both proximal (76 +/- 5 in KHS, 277 +/- 65 in phosphate, 190 +/- 11 in Tris-HEPES, 268 +/- 46 in acetate and 620 +/- 81 microM in lactate) and distal tubules (68 +/- 4 in KHS, 162 +/- 27 in phosphate, 247 +/- 45 in Tris-HEPES, 281 +/- 50 in acetate buffer and 482 +/- 85 microM for lactate). There was a decrease in Vmax for amantadine transport (P < .001) in phosphate, Tris-HEPES and acetate buffers in both proximal (4.83 +/- 0.43 in KHS, 2.38 +/- 0.43 in phosphate, 1.77 +/- 0.31 in Tris-HEPES and 1.47 +/- 0.40 nmol mg protein-1 min-1 in acetate) and distal (4.58 +/- 0.13 for KHS, 1.37 +/- 0.27 for phosphate, 1.82 +/- 0.37 for Tris-HEPES and 1.76 +/- 0.20 nmol mg protein-1 min-1 for acetate) tubules. However, Vmax was not depressed (P < .09) in lactate buffer for proximal (2.87 +/- 0.24 in KHS and 3.12 +/- 0.34 nmol mg protein-1 min-1 in lactate) or distal tubules (2.42 +/- 0.26 in KHS and 3.21 +/- 0.31 nmol mg protein-1 min-1 in lactate), although a slight increase was observed in distal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Amantadine↗
PubMed1987

Measurements of K+-driven Cl- uptake in thylakoids: inhibition of uptake by antibodies raised to the major polypeptides of the Cl(-)-efflux active particle(s).

The mechanism of a K+-driven Cl- accumulation against a concentration gradient was investigated by flow dialysis after addition of K+-Hepes. Non-specific chloride binding, measured in the presence of choline-Hepes, accounted for approximately 50% of the observed uptake in this system. The K+-Hepes-driven Cl- uptake was inhibited by poly-l-lysine and by two antibodies raised to the major polypeptides of the Cl(-)-efflux active particle. Poly-l-lysine had no effect on Cl- binding estimated with choline-Hepes.

Antibodies↗
PubMed2000

Intracellular pH regulation and buffer capacity in CO2/HCO3-buffered media in cultured epithelial cells from rainbow trout gills.

The influence of a CO2/HCO3(-)-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pHi) was monitored by continuous spectrofluorometric recording from cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH = 7.70 were transferred to normal CO2/HCO3(-)-buffered medium ¿PCO2 = 3.71 mmHg, [HCO3-] = 6.1 mmol l(-1), extracellular pH (pHe) = 7.70¿, they exhibited a brief acidosis but subsequently regulated the same pHi (approximately 7.41) as in HEPES. Buffer capacity (beta) increased by the expected amount (5.5-8.0 slykes) based on intracellular [HCO3-], and was unaffected by most drugs and treatments. However, after transfer to high PCO2 = 11.15 mmHg, [HCO3-] = 18.2 mmol l(-1) at the same pHe = 7.70, the final regulated pHi was elevated (approximately 7.53). The rate of correction of alkalosis caused by washout of this high PCO2, high-HCO3- medium was unaffected by removal of extracellular Cl-. Removal of extracellular Na + lowered resting pHi and greatly inhibited the rate of pHi recovery from acidosis. Bafilomycin A1 (3 micromol l(-1)) had no effect on these responses. However amiloride (0.2 mmol l(-1)) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pHi, the latter differing from the response in HEPES where amiloride also lowered resting pHi. Similarly 4-acetamido-4'- isothiocyanatostilbene-2,2'-disulfonic acid, sodium salt (0. 1 mmol l(-1)) did not affect resting pHi but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl(-1) also slowed the rate of recovery but greatly increased beta by an unknown mechanism; when this was taken into account, H+ extrusion rate was unaffected. These results are consistent with the presence of Na+ -(HCO3)N co-transport and/or Na+-dependent HCO3(-)/Cl(-) exchange, in addition to Na+/H+ exchange, as mechanisms contributing to "housekeeping" pHi regulation in gill cells in CO2/HCO3(-) media, whereas only Na+/H+ exchange is seen in HEPES. Both Na(+)-independent Cl-/HCO3(-) exchange and V-type H(+)-ATPase mechanisms appear to be absent from these cells cultured in isotonic media.

Acid-Base Equilibrium↗
PubMed2001

HCO3- potentiates the cAMP-dependent secretory response of the human distal colon through a DIDS-sensitive pathway.

We used the Ussing short-circuit technique to investigate the role of HCO3- in the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretory response of the human distal colon. In HCO3(-)-free 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-Ringer's, forskolin (10 mumol l-1 mucosal and serosal) evoked a sustained increase in short-circuit current (Isc) (delta Isc = 24 +/- 3 microA cm-2, n = 57). However, this was only 25% of the forskolin-stimulated Isc response in HCO3(-)-Ringer's (delta Isc = 84 +/- 8 microA cm-2, n = 57). The reduced response to forskolin in HCO3(-)-free HEPES-Ringer's was not due to inhibition of the secretory mechanism by HEPES, as replacing HCO3- with a different buffer, N-tris[hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES), had a similar effect and inclusion of HEPES in the HCO3(-)-Ringer's did not reduce the secretory response. Similarly, it was not due to an indirect modulation of electrogenic Cl- secretion, as the forskolin-stimulated bumetanide-sensitive Isc was comparable in the two Ringer's. Rather it was due to the activation of a HCO3(-)-dependent Isc which was inhibited by serosal 4,4'-diisothiocyano-stilbene-2,2'-disulphonate (DIDS). This DIDS-sensitive Isc was not inhibited by acetazolamide, but it was inhibited by the replacement of bathing solution Cl- with gluconate, suggesting a role for a Na(+)-dependent Cl-/HCO3- exchanger in the cAMP-dependent secretory response of the human distal colon.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2005

Impaired regulation of pH homeostasis by oxidative stress in rat brain capillary endothelial cells.

(1) Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH(i)) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH(i) regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H2O2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH(i) was monitored by detecting the fluorescent changes. (2) The steady-state pH(i) of RBCE cells in HEPES-buffer (6.83 +/- 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 +/- 0.08). Cells were exposed to NH4CI to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na+ / H+ exchanger, HCO3- / Cl- exchange mechanism is also involved in the restoration of pH(i) after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na+ / H+ exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. (3) No pH(i) recovery was detected when the washout after an intracellular acid load occurred in nominally Na+ -free HEPES-buffered medium or in the presence of 10 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na+ / H+ exchanger. The new steady-state pH(i) were 6.37 +/- 0.02 and 6.60 +/- 0.02, respectively. (4) No detectable change was observed in the steady-state pH(i) in the presence of 100 microM H2O2; however, recovery from NH4Cl prepulse-induced intracellular acid load was inhibited when H2O2 was present in 50 or 100 microM concentration in the HEPES-buffered medium during NH4Cl washout. These data suggest that H2O2 is without effect on the activity of Na+ / H+ exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.

Acidosis↗