HEPES-buffered Ringer Research Articles

Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Intestinal Permeability of Loperamide in Physiological Buffer

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC–MS/MS for the determination of loperamide in HEPES-buffered Ringer's solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d3) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer's solution as a matrix compound.

Metabolic costs induced by lactate in the toad Bufo marinus: new mechanism behind oxygen debt?

The mechanism of an increase in metabolic rate induced by lactate was investigated in the toad Bufo marinus. Oxygen consumption (Vo(2)) was analyzed in fully aerobic animals under hypoxic conditions (7% O(2) in air), accompanied by measurements of catecholamines in the plasma, and was measured in isolated hepatocytes in vitro under normoxia by using specific inhibitors of lactate proton symport [alpha-cyano-4-hydroxycinnamate (CHC)] and sodium proton exchange (EIPA). The rise in metabolic rate in vivo can be elicited by infusions of hyperosmotic (previous findings) or isosmotic sodium lactate solutions (this study). Despite previous findings of reduced metabolic stimulation under the effect of adrenergic blockers, the increase in Vo(2) in vivo was not associated with elevated plasma catecholamine levels, suggesting local release and effect. In addition to the possible in vivo effect via catecholamines, lactate induced a rise in Vo(2) of isolated hepatocytes, depending on the concentration present in a weakly buffered Ringer solution at pH 7.0. No increase was found at higher pH values (7.4 or 7.8) or in HEPES-buffered Ringer solution. Inhibition of the Lac(-)-H(+) transporter with alpha-CHC or of the Na(+)/H(+) exchanger with EIPA prevented the increase in metabolic rate. We conclude that increased Vo(2) at an elevated systemic lactate level may involve catecholamine action, but it is also caused by an increased energy demand of cellular acid-base regulation via stimulation of Na(+)/H(+) exchange and thereby Na(+)-K(+)-ATPase. The effect depends on entry of lactic acid into the cells via lactate proton symport, which is likely favored by low cellular surface pH. We suggest that these energetic costs should also be considered in other physiological phenomena, e.g., when lactate is present during excess, postexercise Vo(2).

Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium.

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.

Effects of bicarbonate versus HEPES buffering on measured properties of neurons in the salamander retina.

Electrophysiological studies of the isolated retina involve perfusing the tissue with a physiological Ringer's. Organic pH buffers such as HEPES have become increasingly popular in recent years because for many purposes they offer a convenient and reliable alternative to the more traditional bicarbonate/CO2. In this paper, however, we report that important functional properties of rods, bipolar cells, and horizontal cells in the salamander, Ambystoma tigrinum, are sensitive to the choice of buffer and, in the case of horizontal cells, that sensitivity is acute. In bicarbonate/CO2 Ringer's, the dark potential of the horizontal cell was typically near -50 mV and saturating light caused it to hyperpolarize to about -75 mV. On switching to HEPES-buffered Ringer's at the same pH, horizontal cells depolarized in darkness to about -20 mV, close to the chloride equilibrium potential, and the kinetics of their light responses changed. The cone-driven components of light responses increased in size relative to rod-driven components. Saturating lights still hyperpolarized the cells to -75 mV, however. Horizontal cells, being coupled via gap junctions, form a syncytium and syncytial length constants, measured in bicarbonate/CO2 Ringer's, were generally in the range 150-225 microm. On switching to HEPES-buffered Ringer's, length constants increased substantially to 250-330 microm. All these changes were reversible. We discuss our findings within the context of the cell's ability to regulate its internal pH.

Secretagogue-induced apocrine secretion in the Harderian gland of the rat.

Harderian glands of male albino rats were stimulated with secretagogues and examined by transmission and scanning electron microscopy for the purpose of studying the apocrine secretory mechanism. Rats in the control group were perfused with standard HEPES-buffered Ringer's solution. Their glandular endpieces showed wide lumina that contained few secretory materials; spontaneous exocytosis was sometimes observed. However, there were no features suggestive of an apocrine secretory mechanism or myoepithelial cell contractions. After stimulation with NaF+AlCl3 or carbachol in HEPES-buffered Ringer's solution, the rats shed "bloody tears" and the glandular lumina were jammed with apical protrusions, cytoplasmic material and secretory products. The basal surface of the glandular cells showed bulging caused by myoepithelial cell contraction. Perfusion with HEPES-buffered Ringer's solution containing papaverine inhibited secretagogue-induced myoepithelial cell contraction but not the enhanced secretory activities of the glandular cells. The present results demonstrate that the Harderian gland of the rat can release secretory material not only by exocytosis, but also by an apocrine mechanism under stimulating conditions, and that myoepithelial cell contraction may not be involved in causing apical protrusion in the glandular cells.

G-protein activation enhances Ca+2-dependent lipid secretion of the rat Harderian gland

We studied the secretory mechanism of the Harderian gland of rats. After perfusion with HEPES-buffered Ringer's solution containing NaF (10 mM) with AlCl3 (10 microM), a G-protein activator, the glandular cells of the Harderian gland showed massive exocytosis and apocrine-like protrusions on the luminal surface. Some of the secretory vacuoles aggregated within the cytoplasm, and large vacuoles were formed. Contraction of the myoepithelial cells covering the glandular endpieces caused a narrowing of the glandular lumina, which contained cytoplasmic fragments, and deformation of the basal contour of the glandular end-pieces. The basal regions of the glandular cells also bulged between the myoepithelial cells. Secretory vacuoles were also discharged to the lateral cell surface, and the intercellular spaces were dilated. The enhanced secretory activities of the glandular cells and the contraction of the myoepithelial cells were similar to those in rats stimulated with 10 microM carbachol (CCh). However, dilatation of the endoplasmic reticulum in glandular cells (type A cells), which leads to the formation of small vesicles, was observed in those glands stimulated by NaF+AlCl3, but not in those stimulated by CCh. Removal of Ca2+ from the perfusing HR or addition of EDTA (0.5 mM) diminished and inhibited NaF+AlCl3- or CCh-enhanced secretory activity of the glandular cells and also allayed the deformation of glandular cells caused by myoepithelial cell contraction. The present results demonstrate the involvement of G-proteins and Ca(2+)-influx in the lipid secretion of glandular cells and in the contraction of myoepithelial cells of the Harderian gland in rats.

Ligand-gated currents of alpha and beta ganglion cells in the cat retinal slice

1. We studied the receptor pharmacology of the ligand-gated currents of ON- and OFF- alpha and beta ganglion cells in a cat retinal slice preparation using the whole cell recording variation of the patch-clamp technique. Cat retinal slices were cut in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer and incubated in a bicarbonate-buffered solution. Ganglion cells were voltage clamped at -70 mV in HEPES-buffered Ringer solution. The pipette solution contained a low concentration of Cl- to distinguish mixed cationic from Cl(-)-mediated conductances, and Lucifer yellow (0.5%) was included for identification of the cell type. 2. In Ringer solution containing 1.2 mM Mg2+, current-voltage (I-V) curves of responses to the excitatory amino acid agonist (EAA) N-methyl-D-aspartate (NMDA) (200 microM) revealed a J-shaped function. In Mg(2+)-free Ringer solution containing 200 microM Cd2+ to block synaptic transmission, NMDA (200 microM) elicited an inward current 5-8 times larger at -70 mV. In both conditions I-V curves of the NMDA-induced currents reversed near 0 mV. These results suggest that there are NMDA EAA receptors present directly on the dendrites of alpha and beta ganglion cells. Responses to NMDA were blocked by +/- 2-amino-7-phosphonoheptanoic acid (AP7) (200 microM). 3. In Ringer solution containing 200-1,000 microM Cd2+ to block synaptic transmission, both ON- and OFF- alpha and beta cells responded to kainic acid (10-50 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) (20-70 microM), and quisqualic acid (0.1-30 microM) with inward currents that reversed near 0 mV. These responses were blocked by the quinoxaline EAA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM). The metabotropic agonists 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (25 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) in the presence of Cd2+ evoked little or no response for all cells tested. 4. In the presence of Cd2+, alpha and beta cells responded to gamma-amino-butyric acid (GABA) (200 microM) and glycine (200 microM) with inward currents that reversed near -35 mV, the calculated chloride equilibrium potential Ecl. Responses to GABA and glycine were both strongly desensitizing. (+)Bicuculline methyl chloride (20 microM) blocked an average of 90% of the inward current evoked by 200 microM GABA on all ganglion cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Model of bicarbonate secretion by resting frog stomach fundus mucosa. I. Transepithelial measurements.

In the present in vitro experiments on gastric fundus mucosa of Rana esculenta we try to define the mechanism of alkaline secretion that is observed in summer frogs in the resting stomach (blockage of HCl secretion by ranitidine, 10(-5) mol/l). The transepithelial voltage and the rate of alkalinization (ASR) of an unbuffered gastric lumen perfusate was measured as a function of serosal (and mucosal) fluid composition. ASR was high (0.88 +/- S.E. 0.09 microEq.cm-2.h-1, n = 11) during serosal bath perfusion with HCO(3-)-Ringer solution, decreased slightly to 0.50 +/- 0.07 microEq.cm-2.h-1 (n = 6) in HCO(3-)-free HEPES-buffered Ringer solution of the same pH, and decreased to approximately 20% when carbonic anhydrase was inhibited by acetazolamide. While replacement of mucosal or serosal Cl- did not--within 1 h--significantly alter ASR, replacement of serosal Na+ in the presence or absence of HCO3- strongly reduced ASR, and a similar reduction was observed after serosal application of the anion transport inhibitor DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonate, 2.10(-4) mol/l), the metabolic poison rotenone (10(-5) mol/l), the uncoupler dinitrophenol (10(-4) mol/l), and the Na+ pump inhibitor ouabain (10(-4) mol/l), while serosal amiloride (10(-4) mol/l) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

<b>MORPHOLOGICAL CHANGES IN PANCREATIC ACINAR CELLS CAUSED BY SUPRAMAXIMAL CHOLECYSTOKININ </b><b>STIMULATION </b>

Rat pancreatic acini were isolated by collagenase digestion and exposed to cholecystokinin octapeptide (CCK-8) at concentrations of 0, 10 -10 and 10 -8 M. Secretion was monitored by measuring the release of amylase activity into the incubation medium that consisted of oxygenated HEPES-buffered Ringer's solution containing 2.5 mM Ca 2+ . The release of lactate dehydrogenase (LDH) into the incubation medium was measured to detect cell injury. The morphology of acinar cells was studied by light microscopy and scanning and transmission electron microscopy. A marked secretory response to stimulation with an optimal concentration of CCK-8 (10 -10 M) resulted in unaltered acinar cell morphology and in insignificantly increased LDH release as compared to non-stimulated controls

Separation of the Rabbit Ciliary Body Epithelial Layers in Viable Form: Identification of Differences in Bicarbonate Transport

Sections of whole ciliary (CB) dissected from Dutch belted rabbits were incubated for 2 hr at 36°C in a 30μM Ca 2+ Ringer's. This incubation resulted in the spontaneous dissociation of the two cell layers comprising this epithelium, each remaining firmly cohesive with its own basement membrane. The inner limiting membrane with its adherent non-pigmented epithelium (NPE) was then mechanically removed from the surface exposing the apical surface of the pigmented epithelium (PE). Ultrastructural examination revealed no noxious effects in most cells although gross morphological changes in the NPE cells were noted. The newly separated layers were loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. Most cells of both layers acquired stable BCECF fluorescence indicating viability. To achieve a preliminary evaluation of differences in PE and NPE bicarbonate transport, dye-loaded tissues were perfused in a flow-through chamber which was mounted on a microscope equipped for quantitative epifluorescence. The intracellular pH (pH i) of groups of cells (5-10) was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm. In Hepes-buffered Ringer's the pH is for the PE and NPE were 7·20±0·10 and 7·33±0·14 (±S.D., n = 6), respectively. Replacement of 28 mM Hepes by 28 mM HCO 3- /5% CO 2 led to a 0·13 pH i increase in the PE and a decrease of 0·27 U in the NPE. The pH i responses of the two cell layers to removal and/or reintroduction of Na + and/or Cl - were also highly dissimilar. These observations suggest that each cell type is endowed with distinct mechanisms of bicarbonate transport.

Corneal tolerance of vitrifiable concentrations of glycerol

Equilibration of corneas with sufficiently high concentrations of cryoprotectants to inhibit potentially damaging ice formation during cryopreservation has not yet been achieved. This study examined the effects on the structure and function of rabbit corneal endothelium of the low toxicity cryoprotectant glycerol. Corneas were exposed to concentrations ranging from 2.0 to 6.8 M glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 m M sodium bicarbonate and 6% w v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 °C for 6 h. Endothelial structure was observed using specular microscopy during perfusion and scanning electron microscopy after perfusion. Corneas tolerated exposure to 2.0 and 3.4 M glycerol for 20 min at 4 and −5 °C, respectively. Tolerance of 4.8 M glycerol for 10 min at −10 °C was improved by decreasing the dilution temperatures. Ten-minute exposure to 6.1 and 6.8 M glycerol was tolerated at −15 °C. In all cases corneas initially showed signs of damage but endothelial function was regained following structural repair. Corneas exposed to 6.8 M glycerol and cooled below the glass transition temperature were nonfunctional after warming. Ice formation during warming was believed to be the cause of injury.

The potential of an equimolar combination of propane-1,2-diol and glycerol as a vitrification solution for corneas

Corneas must first be equilibrated with multimolar concentrations of cryoprotectants if the formation of ice during cryopreservation is to be avoided by vitrification at practicable cooling rates. Rabbit corneas were exposed to equimolar mixtures of the cryoprotectants propane-1,2-diol and glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w v bovine serum albumin. Endothelial function was assessed by monitoring its ability to control stromal hydration during perfusion of the endothelial surface at 34 °C for 6 h. Endothelial morphology was observed by specular microscopy during perfusion and by scanning electron microscopy after perfusion. Endothelial pump activity and structural integrity of the endothelial layer were demonstrated after 20 min exposure at 4 °C to a total concentration of 1.4 mol/liter cryoprotectant (i.e., 0.7 mol/liter propane-1,2-diol + 0.7 mol/liter glycerol). Exposure to 2.0 and 3.4 mol/liter cryoprotectant for 20 min at 4 ° and − 5 °C, respectively, resulted in initial endothelial damage, but this repaired and a functioning endothelial pump was subsequently demonstrated. Although exposure to 4.1 mol/ liter cryoprotectant for 10 min at − 10 °C caused irreparable damage to 2 4 corneas, reduced dilution temperatures together with increased dilution time allowed exposure to 4.8 and 5.5 mol/liter cryoprotectant with retention of endothelial pump activity. Exposure to 6.1 mol/liter cryoprotectant for 10 min at − 15 °C caused endothelial damage which was not mitigated by the presence of 2.5% w v chondroitin sulfate. Endothelial function may be improved by further modification of addition and dilution protocols or by exposure to the cryoprotectants at lower temperatures.

Corneal tolerance of vitrifiable concentrations of propane-1,2-diol

The merit of corneal cryopreservation by vitrification as opposed to conventional freezing is the avoidance of ice damage which is believed to disrupt the integrity of the corneal endothelium resulting in loss of corneal transparency. The cornea must be equilibrated with high concentrations of cryoprotectant in order to achieve vitrification at practicable cooling rates. In an earlier study, corneas were exposed to 3.4 mol/liter propane-1,2-diol (Rich and Armitage (1990) Cryobiology 27, 42–54) . The present study exposed rabbit corneas to concentrations of propane-1,2-diol between 3.4 and 5.4 mol/liter in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, 6% ( w v ) bovine serum albumin, and 2.5% ( w v ) dextran sulfate. Dextran sulfate was as effective as chondroitin sulfate at improving endothelial tolerance of 3.4 mol/liter propane-1,2-diol. This beneficial effect may be linked to the polyanionic nature of these molecules. Corneas exposed to 5.4 mol/liter propane-1,2-diol were cooled in liquid nitrogen vapor at a temperature of −140 °C for 2 h. Warming was achieved by direct transfer to a dilution solution at −10 °C. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 °C for 6 h. Endothelial structure was observed by specular microscopy during the perfusion and by scanning electron microscopy after perfusion. Corneas tolerated exposure to 3.4 mol/liter propane-1,2-diol for 20 min at 0 °C and to 4.1 mol/liter for 10 min at −10 °C. Exposure to 4.8 and 5.4 mol/liter for 10 min at −10 °C caused endothelial damage, although a degree of endothelial function was retained. Function following exposure to 5.4 mol/liter was improved by reducing the temperature of exposure to −15 °C. Corneas cooled after exposure to 5.4 mol/liter propane-1,2-diol for 10 min at −15 °C apparently vitrified, but devitrified on warming. The corneas swelled to such an extent during perfusion that the endothelium could not be viewed by specular microscopy, subsequent scanning electron microscopy showed a severely disrupted endothelium.

Further studies of electrogenic Na+/HCO cotransport in glial cells of necturus optic nerve: Regulation of pHi

In the presence of Ba++, an increase in the bath HCO3- at constant CO2 (i.e., variable bath pH) produced a hyperpolarization. The hyperpolarizing effect of adding HCO3-/CO2 at constant bath pH was not significantly affected by the presence of 50 mumol/l strophanthidin. In the absence of Ba++, addition of HCO3-/CO2 at constant bath pH produced a Na(+)-dependent hyperpolarization. Therefore, CO2 movements, electrogenic Na+/K+ pump activity and changes in Ba++ binding do not contribute significantly to the hyperpolarization induced by HCO3-. These results along with the results of previous studies (Astion et al: J Gen Physiol 93:731, 1989) strongly suggest that the hyperpolarization induced by the addition of HCO3- is due to an electrogenic Na+/HCO3- cotransporter, which transports Na+, HCO3- (or its equivalent), and net negative charge across the glial membrane. To study the role of electrogenic Na+/HCO3- cotransport in the regulation of pHi in glial cells, we used intracellular double-barreled, pH-sensitive microelectrodes. At a bath pH of 7.5, the mean initial intracellular pH (pHi) was 7.32 (SD 0.03, n = 6) in HEPES-buffered Ringer's solution and 7.39 (SD 0.1, n = 6) in HCO3-/CO2 buffered solution. These values for pHi are more than 1.2 pH units alkaline to the pHi predicted from a passive distribution of protons; thus, these cells actively regulate pHi. Superfusion and withdrawal of 15 mmol/l NH4+ induced an acidification of 0.2 to 0.3 pH units, which recovered toward the original steady-state pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Propane-1,2-diol as a potential component of a vitrification solution for corneas

Any method of cryopreservation of the cornea must maintain integrity of the corneal endothelium, a monolayer of cells on the inner surface of the cornea that controls corneal hydration and keeps the cornea thin and transparent. During freezing, the formation of ice damages the endothelium, and vitrification has been suggested as a means of achieving ice-free cryopreservation of the cornea. To achieve vitrification at practicable cooling rates, tissues must be equilibrated with high concentrations of cryoprotectants. In this study, the effects of propane-1,2-diol on the structure and function of rabbit corneal endothelium were studied. Corneas were exposed to concentrations of propane-1,2-diol ranging from 10 to 30% v/v in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 °C for 6 hr. Exposure to 10–15% v/v propane-1,2-diol was well tolerated for 20 min at 4 °C when the cryoprotectant was removed in steps or by sucrose dilution. However, exposure to 25% v/v propane-1,2-diol for 20 min at 0 or −5 °C was consistently tolerated only when 2.5% w/v chondroitin sulfate was included in the svehicle solution. Exposure to 30% v/v propane-1,2-diol was harmful at −5 and −10 °C. The endothelial damage following exposure to 30% v/v propane-1,2-diol was probably the result of a toxic effect rather than osmotic stress. Although 25% v/v propane-1,2-diol does not vitrify at cooling rates that are practicable for corneas, it could at this concentration form a major component of a vitrification solution comprising a mixture of cryoprotectants.

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 HCO 3 − -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 70ms. Kynurenic acid (an excitatory amino acid antagonist), or Mn 2+, 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 pH o and extracellular volume transients was studied using tetramethylammonium as an extracellular volume indicator. In nominally HCO 3 −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 Mn 2+, 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.

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 ~ 7.1 in a HEPES-buffered, HCO3-free Ringer's solution. Average pHi in Necturus cells in CO2/HCO3-buffered Ringer's solution was ~7.0. An ammonium-loading technique was used to acidify the cell to measure intrinsic buffering capacity (βi) and to monitor pHi recovery. Average βi = 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 μM 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.

Anomalous 32P-labelling of human erythrocytes during prolonged incubations in vitro

Conference Article| December 01 1987 Anomalous 32P-labelling of human erythrocytes during prolonged incubations in vitro GRAHAM J. KEMP; GRAHAM J. KEMP 1Department of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, U.K. Search for other works by this author on: This Site PubMed Google Scholar DJAMEL KHODJA; DJAMEL KHODJA 1Department of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, U.K. Search for other works by this author on: This Site PubMed Google Scholar ALAN BEVINGTON; ALAN BEVINGTON 1Department of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, U.K. Search for other works by this author on: This Site PubMed Google Scholar R. GRAHAM; R. GRAHAM 1Department of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, U.K. Search for other works by this author on: This Site PubMed Google Scholar G. RUSSELL G. RUSSELL 1Department of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (6): 1141–1142. https://doi.org/10.1042/bst0151141 Article history Received: June 18 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation GRAHAM J. KEMP, DJAMEL KHODJA, ALAN BEVINGTON, R. GRAHAM, G. RUSSELL; Anomalous 32P-labelling of human erythrocytes during prolonged incubations in vitro. Biochem Soc Trans 1 December 1987; 15 (6): 1141–1142. doi: https://doi.org/10.1042/bst0151141 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ATPase, adenosine 5′-triphosphatase, HR, Hepes-buffered Ringer solution, OP, organic phosphates, Pi, orthophosphate, PCV, packed cell volume, G3PDH, glyeraldehyde-3-phosphate dehydrogenase This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

Activation of luminal Na+/H+ exchange in distal nephron of frog kidney. An early response to aldosterone.

Increased chronic intake of K+ induces H+ and K+ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed) Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pHlu) was measured continuously with pH-sensitive microelectrodes while aldosterone (3 X 10(-7) to 3 X 10(-6) mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (Vte) from 8.5 +/- 1.1 mV to 4.0 +/- 0.6 mV pHlu dropped from 7.73 +/- 0.02 to a new steady-state value of 7.17 +/- 0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10(-3) mol/l amiloride reversed luminal acidification to a pHlu of 7.68 +/- 0.04; at the same time Vte recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone--after interaction with cytoplasmic receptors--activates the luminal, amiloride-inhibitable Na+/H+ exchanger. This mechanism could explain enhanced H+ secretion found in the K+ adapted animal.

Intracellular mediators of Na+-K+ pump activity in guinea pig pancreatic acinar cells

The involvement of Ca2+ and cyclic nucleotides in neurohormonal regulation of Na+-K+-ATPase (Na+-K+ pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by [3H]ouabain binding and by ouabain-sensitive 86Rb+ uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of [3H]ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in Ca2+-free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular Ca2+, while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free Ca2+ levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent Ca2+ chelator. Basal intracellular Ca2+ concentration ([Ca2+]i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively. Forskolin, secretin, and cAMP analogues had no effect on [Ca2+]i, nor did they either reduce or potentiate the rise in [Ca2+]i evoked by CCh.(ABSTRACT TRUNCATED AT 250 WORDS)