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PubMed1996

Chloride currents in skeletal muscles of Bufo arenarum.

C1- currents (ICl) were measured in short fibers (1-2 mm) from the lumbricalis muscle of toads (Bufo arenarum) with two microelectrodes (15 degrees C). Initially the fibers were equilibrated in a high (K+)-containing solution: (mM) K2SO4 68; Na2SO4 20; KCl 60; CaSO4 8; MgSO4 1; HEPES 2.5. Constant pulses were applied when all the external K+ was replaced by Cs+: Cs2SO4 68; Na2SO4 20; CsCl 60; CaSO4 8; HEPES 2.5 (pH 7.5). Under these conditions about 80-90% of the current is carried by Cl-. The current-voltage relation is almost linear implying constant conductance and hence voltage-independent permeability. The voltage dependence of the net C1- current could be fitted by constant field equation with a PCl of 3.3 x 10-6 cm/sec. In a separate group of experiments a two-pulse technique was used to estimate the availability and the inactivation of the initial ICl during a test pulse. After returning the potential to the holding potential for various times, test pulses of the same amplitude and duration of the prepulses were applied. The initial current during the test pulse was 70% of the initial current during the prepulse and the recovery was complete in less than 300 msec with a linear relationship between the current during the test pulse and the amplitude of the preceding prepulse. When the test pulses were preceded by a positive prepulse, the initial current for any given test pulse was larger than with a negative prepulse. If we assumed that the initial current during the test pulse is a measure of the number of channels open at the end of the prepulse, these results suggest that hyperpolarizing pulses inactivate and depolarizing prepulses activate the ICl.

Animals↗

PubMed1996

Chloride currents activated by calcitonin and cAMP in primary cultures of rabbit distal convoluted tubule.

Chloride (Cl-) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33 degrees C) contained (in mM): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-D-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl- current activated by 10(-5) M forskolin, 10(-3) M 8-bromo adenosine 3',5'-cyclic monophophosphate (8 Br-cAMP), 10(-6) M phorbol 12-myristate 13-acetate (PMA), 10(-3) M intracellular adenosine 3',5'-cyclic monophophosphate (cAMP) and 10(-7) M calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br- > Cl- > > I- > glutamate. This current was inhibited by 10(-3) M diphenylamine-2-carboxylate (DPC) and 10(-4) M 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl- current blocked by DIDS. To determine the exact location of the Cl- conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mM SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl- removal from the apical solution induced a Cl- efflux which was stimulated by 10(-5) M forskolin, 10(-7) calcitonin and inhibited by 10(-5) M NPPB. In 140 mM NaBr, forskolin stimulated an apical Br- influx through the Cl- pathway. Forskolin and calcitonin had no effect on the basolateral Cl- permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl- conductance in the apical membrane through a cAMP-dependent mechanism.

Animals↗

PubMed1999

Transient electric birefringence of human erythroid spectrin dimers and tetramers at ionic strengths of 4 mM and 53 mM.

In conventional electrooptic studies the sample ionic strength must for technical reasons be kept below about 3 mM, which is only 2% of the ionic strength at physiological conditions. In particular for flexible polyelectrolytic macromolecules it can in general not be ruled out that both the conformational average and dynamics at ionic strength 3 mM and below may differ significantly from what it is at physiological conditions. Here we report on the first electrooptic study of human erythroid spectrin dimers and tetramers at ionic strengths higher than 3 mM. All measurements in this study were carried out at both ionic strength 4 mM (2.5 mM HEPES + 1 mM NaCl) and 53 mM (2.5 mM HEPES + 50 mM NaCl). Spectrin tetramers were studied only at 4 degrees C whereas the dimers were studied at both 4 degrees C and 37 degrees C. At 4 degrees C there is a striking quantitative similarity between the transient electric bire-fringence (TEB) of spectrin dimers and tetramers. Also, the TEB of spectrin dimers at 37 degrees C was very similar to the results at 4 degrees C. The contour length and the molecular weight of spectrin dimers and tetramers are known. The dominating TEB relaxation time is in all cases only a fraction of what is predicted theoretically if the spectrin dimers and tetramers are assumed to be stiff and extended molecules. In sum, the new TEB data constitute strong electrooptic evidence confirming that spectrin dimers and tetramers have a highly flexible structure, and demonstrate for the first time that a major part of the intrachain dynamics of the spectrin is quite insensitive to an increase of the ionic strength from 4 mM to 53 mM. Use of the reversing electric field pulse technique for all conditions studied yields TEB data suggesting that the orientation of both spectrin dimers and tetramers in an electric field is dominated by a permanent rather than an induced electric dipole moment.

Biophysical Phenomena↗

PubMed2003

Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent.

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.

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

PubMed1998

Role of physiological HCO3-buffer on intracellular pH and histamine release in rat peritoneal mast cells.

The purpose of this study was to examine how intracellular pH (pHi) regulation and histamine release are affected by HCO3- in rat peritoneal mast cells. The pHi was measured using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We observed a pHi of 6.88+/-0.012 (n=24) in resting mast cells exposed to a HEPES buffer (pH 7.4), but a sustained drop of 0.21 pH units to 6.67+/-0.015 (n=23) when we exposed the mast cells to a HEPES/HCO3- buffer equilibrated at all time with 5% CO2 (pH 7.4). This fall in pHi is inhibited by the carbonic anhydrase inhibitor dichlorphenamide and is Na+-independent, indicating the involvement of Na+-independent Cl-/HCO3- exchange activity. Furthermore removal of external Cl- in the presence but not in the absence of HCO3- reversed the Cl-/HCO3- exchange and induced an alkaline load. The recovery from this alkaline load was dependent on external Cl- but independent of Na+. Both the alkalinization and the recovery were inhibited by the anion transport inhibitor 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In addition, 36Cl- uptake measurements confirm the presence of a Cl-/HCO3- exchanger. Histamine release stimulated by antigen and compound 48/80 was substantially reduced in the presence of HEPES/ HCO3- buffer (pHo 7.4, pHi 6.66). Histamine release was increased, however, when pHi was clamped to 6.66 in HCO3--free media (pHo 6.9). We conclude that: (1) Na+-independent Cl-/HCO3- exchange determines steady-state pHi in rat peritoneal mast cells; and (2) the reduction in histamine release observed in the presence of HCO3- is not due to its effect on pHi per se, but rather on other changes in ion transport.

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

PubMed2003

Synthesis, structure analysis, solution chemistry, and in vitro insulinomimetic activity of novel oxovanadium(IV) complexes with tripodal ligands containing an imidazole group derived from amino acids.

Structures, chemical properties, and in vitro insulinomimetic activities of new vanadyl [oxovanadium(IV), VO(2+)] complexes with five tripodal ligands containing an imidazole functionality were examined. The ligands, N-(carboxymethyl)- N-(4-imidazolylmethyl)amino acids, contain glycine, ( S)- and ( R)-alanine, and ( S)- and ( R)-leucine residues. The molecular structures of the latter four alanine- and leucine-containing complexes were determined by X-ray analysis. The coordination geometry around each vanadium center was octahedral, where an imino nitrogen occupied the apical site and two carboxylate oxygens, an imidazole nitrogen, and a water molecule coordinated in the equatorial plane. The spectroscopic properties of the complexes were characterized by means of IR, electronic absorption, and CD spectra. Acid dissociation constants (p K(a)) and protonation sites of the ligands were determined by a combination of potentiometric titrations and (1)H NMR spectra. The potentiometric study demonstrated that stability constants (log beta) were not so different among the present complexes (14.0-14.9) and a species of molecular complex with a 1:1 metal:ligand ratio existed predominantly at physiological pH 7.4. EPR parameters indicated that the species at pH 7.4 had an octahedral structure similar to the complex in the solid state. On the other hand, an EPR study in phosphate buffer (pH 7.4) suggested that inorganic phosphate coordinated to the vanadium center instead of the imidazole group in the presence of excess phosphate ion. Cyclic voltammograms in the phosphate buffer showed chemically reversible oxidation waves, whereas irreversible oxidation waves were observed in non-coordinating HEPES buffer. Moreover, the oxidation potential of each complex in phosphate buffer was more positive than that in HEPES buffer. Partition coefficients of the present complexes in a n-octanol/saline system were very low, probably due to hydrophilicity of the imidazole group. The in vitro insulinomimetic activities were estimated on the basis of the ability of the complexes to inhibit epinephrine-stimulated free fatty acid release from isolated rat adipocytes. The achiral glycine-derivative complex exhibited the highest insulinomimetic activity, which was higher than that of VOSO(4) as a positive control. Putting our previous observations together, it was found that the vanadyl complexes with tetradentate amino acid derivatives having no alkyl side chain tend to have high in vitro insulinomimetic activity.

Alanine↗

PubMedDate not supplied

Effects of iron chelates on the transferrin-free culture of rat dermal fibroblasts through active oxygen generation.

Effects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 micrograms/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 microM FeSO4 in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H2O2) generated by superoxide dismutation does not. GG significantly reduced H2O2 cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H2O2 was added to the medium. FeSO4 and FeCl3 (50 to 100 microM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO4 and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO4 and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe3+ and Fe2+, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.

Animals↗

PubMed1985

Effects of 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid and amiloride on salivary secretion by isolated, perfused rat submandibular glands.

Isolated submandibular glands of adult rats were perfused through the arterial system with oxygenated, HCO3-containing or HCO3-free physiological salt solutions. Secretion of saliva was then induced with acetylcholine (10(-6) M) in the absence or presence of the ion-transport inhibitors 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS), furosemide or amiloride. In HCO3-containing perfusates, 10(-4) M DIDS enhanced the initial secretory response (maximum rate of flow increased 18 per cent), but reduced the overall volume of saliva secreted in a 60-min period by 47 per cent. Furosemide (10(-3) M) alone reduced the volume of saliva by 73 per cent and, in combination with 10(-4) M DIDS, by 82 per cent. Amiloride (10(-3) M) caused a 68 per cent reduction in salivary volumes. Replacement of perfusate HCO3 with HEPES did not affect acetylcholine-induced secretion but enhanced the effects of the transport inhibitors, so that total volume of secretion was reduced 94 per cent by furosemide, 55 per cent by DIDS and 80 per cent by amiloride. In HCO3-containing perfusates, DIDS caused a 30-50 per cent increase in salivary Na+ and residual anion (Na + K - Cl) concentrations but amiloride induced a marked increase in salivary Na+ and Cl- concentrations and a decrease in salivary K+ concentrations. Furosemide caused a marked decrease in salivary Cl- concentrations and a marked increase in residual anions. These effects were similar but of smaller magnitude in HCO3-free, HEPES-containing perfusates.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed1989

Inhibition of 36Cl uptake by stilbene sulphonic acid derivatives and loop diuretics in rat submandibular salivary acini.

Acini were isolated from submandibular glands by enzymatic digestion and incubated in HCO3-containing and HCO3-free [hydroxyethyl-1-piperazine-ethane sulphonic acid (HEPES) buffered] solutions to compare the accumulation of the isotopic tracer 36Cl in the absence and presence of stilbene sulphonic acid derivatives and of loop diuretics. Tracer accumulation was similar in both solutions in the absence of inhibitors and reached 17-19 nmol/mg protein after 5 min of incubation. Exposure to 10(-3) M 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) or to 10(-4) M bumetanide in HCO3-buffered medium resulted in significant reductions in both the initial phase (from 0 to 5 min) and the steady-state phase (5-30 min) of 36Cl accumulation. By contrast, in HCO3-free, HEPES-buffered incubation solution, DIDS was significantly less effective in reducing 36Cl accumulation, while bumetanide was somewhat more effective than in HCO3-buffered medium. At a higher dose (10(-3) M), furosemide was less effective than bumetanide in reducing the steady-state tracer content of the cells in the HCO3-buffered medium (22% versus 35% reduction), although the effects were similar in the initial phase of tracer uptake. Exposure to 10(-4) M 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) caused an increase in the steady-state 36Cl content of the acini of 24 +/- 9%. Exposure to bumetanide and DIDS caused an additive reduction of tracer content, while exposure to bumetanide and SITS resulted in a smaller reduction than with bumetanide alone.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed1991

Degradation of bovine incisor root collagen in an in vitro caries model.

The effects of pH, ionic strength and proteinases on the destruction of bovine incisor root collagen were studied. Experiments were done with powdered and intact root specimens. Completely demineralized root powder was subjected to solutions of varying pH and ionic strength: (a) 0.1 M acetic acid, pH 4.0, (b) 0.1 M acetic acid + 0.15 M KCl, pH 4.0, (c) 0.1 M Hepes, pH 7.0 or to (d) 0.1 M Hepes + 0.15 M KCl, pH 7.0 at 37 degrees C. The surfaces of intact root specimens were exposed to 0.1 M acetic acid, pH 4.0 (which resulted in erosive lesions) or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0 (which produced subsurface lesions) at 37 degrees C. After incubation, the extracts were analysed for soluble collagen and the insoluble matrices were treated with trypsin at 15 degrees C to determine the denatured collagen. To estimate sensitivity to non-specific proteinases, demineralized root powder was also treated with trypsin under physiological conditions of temperature, pH and ionic strength. The denaturation and subsequent solubilization of collagen material from the fibrils could be influenced by variations in pH and ionic strength but these effects were small when compared to proteolytic degradation under physiological conditions. This supports the hypothesis that, in root caries, destruction of exposed matrix collagen depends largely on the presence and activity of proteinases.

Acetates↗

PubMed1985

Differential interaction of cations with the thiamine and biotin transport proteins of Lactobacillus casei.

Lactobacillus casei cells contain separate and specific binding proteins which mediate the cellular uptake of thiamine and biotin. In buffered salt solutions, these proteins exhibit a very high affinity for their vitamin substrate. Dissociation constants (Kd values) at pH 7.5 are 0.03 and 0.15 nM for thiamine and biotin, respectively. Optimal binding of biotin requires the presence of cations. This cation dependence is substantial since the Kd for biotin is 60-fold higher in a buffer containing 0.1 mM K-Hepes, compared with a buffer composed of 50 mM K-Hepes and 5 mM MgCl2. Measurements of Kd versus cation concentration showed that Mg2+ is 300-fold more effective than K+ in promoting biotin binding. The extent of cation dependence decreases as the pH is reduced from 7.5 to 5.0, suggesting that protons can partially fulfill the cation requirement. In contrast, binding of thiamine to the thiamine transport protein shows no dependence on the ionic composition of the medium. These results suggest that the transport protein for the anionic vitamin, biotin, contains a binding site for cations. Cotransport of both the vitamin and cation into the cell might then occur during the normal transport cycle, allowing the cellular uptake of the vitamin to occur against the membrane potential. Conversely, the cationic vitamin, thiamine, does not appear to be transported into the cell as a complex with other ions.

Biotin↗

PubMed1993

Deregulation by zinc of the sodium efflux in barnacle muscle fibers.

Single muscle fibers from the barnacle Balanus nubilus were employed to study the behavior of the resting Na+ efflux toward external and internal application of zinc (Zn2+). This involved both unpoisoned and ouabain-poisoned fibers. The results obtained are as follows: (i) External application of Zn2+, e.g., 2 mM (a maximal dosage) in 10 mM Hepes-ASW (pH 7.3) causes a fall in the resting Na+ efflux which exceeds that caused by 10(-4) M ouabain in companion controls. (ii) The buffer of choice is found to be Hepes, rather than HCO3- or imidazole. (iii) The observed fall in the resting Na+ efflux caused by external application of Zn2+ is concentration-dependent, the IC50 being 10 microM. (iv) The inhibitory effect of Zn2+ is partially reversible; occasionally, however, reversibility is not seen. (v) The Zn(2+)-insensitive component of the Na+ efflux is reduced by 10(-4) M ouabain. (vi) The ouabain-insensitive component of the Na+ efflux is reduced by external application of Zn2+. This response is concentration-dependent. (vii) Preinjection of EGTA reduces the sensitivity of the Na+ efflux to external application of Zn2+. This is true of both unpoisoned and ouabain-poisoned fibers. (viii) (a) The resting Na+ efflux is reduced by injecting Zn2+. Ouabain application reduces the remaining Na+ efflux. (b) Injection of Zn2+ reduces the ouabain-insensitive component of the Na+ efflux. (c) External application of Zn2+ following the injection of Zn2+ reduces the remaining Na+ efflux. Ouabain is ineffective when applied after both maneuvers. (d) Injection of Zn2+ after its external application is without effect. Subsequent application of ouabain is also without effect. (e) Injection or external application of Zn2+ reduces the ouabain-insensitive Na+ efflux. Whereas in the former case subsequent external application of Zn2+ reduces the remaining Na+ efflux, in the latter case Zn2+ injection after external application of Zn2+ is ineffective. Collectively, these results provide evidence in support of the hypothesis that Zn2+ is a potent inhibitor of the ouabain-sensitive and ouabain-insensitive components of the Na+ efflux, and that the inhibitory effect is partly due to the entry of Zn2+ into the myoplasm. They also raise the possibility that the inhibitory effect caused by Zn2+ injection may be the result of Zn2+ leakage from the fiber interior.

Animals↗

PubMed1975

Induced changes in the electron paramagnetic resonance spectra of mammalian catalases.

The EPR spectra of bovine liver catalase, rat liver catalase and human erythrocyte catalase have been measured at 9.0 degrees K. In N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) and Tris buffers at pH 7.0, the liver catalases show EPR spectra typical of rhombically distorted high spin ferric heme with major lines at g = 6.50, 5.35, 1.98. A number of extra lines are also seen; these are weak or absent in human erythrocyte catalase. The effect of the addition of formate, nitrite, acetate, fluoride, azide, hypophosphite and of inactivation with 3-amino-1,2,4-triazole on the degree of rhombic distortion has been studied. There is a good correlation between the low temperature EPR and room temperature optical changes for the binding of formic acid in HEPES and Tris. There is no evidence from EPR spectra for the presence of heme-heme interactions in the binding of formic acid to human erythrocyte catalase. The properties of catalase are altered in phosphate and in distilled water. This is a consequence of the low temperature of measurement.

Amitrole↗

PubMed1989

Identification of calcium conducting channels in isolated boar sperm plasma membranes.

Ion channel recordings were obtained from liposomes containing purified boar sperm plasma membrane proteins using a tip-dip method. Liposomes prepared in HEPES-TRIS and clamped by electrodes containing Ba-HEPES displayed channel activity that was partially inhibited by verapamil or nitrendipine and completely inhibited by La3+. Reversal of current at pipette negative voltages was observed only when Ba2+ ions were also present in the bath solution. These data indicate that channels capable of carrying calcium currents are prominent components of the plasma membrane of mammalian sperm.

Animals↗

PubMed1988

Characterization of midazolam metabolism using human hepatic microsomal fractions and hepatocytes in suspension obtained by perfusing whole human livers.

Isolated human hepatocytes provide a useful model for studying xenobiotic metabolism. However, in vitro studies using human hepatocytes are scarce due to the limited availability of this material. A new methodology is described for obtaining hepatocytes from a whole adult human liver. This procedure is based on (i) the rapid and intense in situ washing step of the organ with Eurocollins then glucose supplemented HEPES buffer (10 mM, pH 7.4) at 4 degrees in order to both minimize the warm ischemic period and remove erythrocytes, and (ii) a perfusion of collagenase solution (0.05% in 10 mM HEPES buffer at 37 degrees) throughout the portal vein according to a recirculated model. All perfused buffers are oxygenized. Hepatocyte viability averaged 85% as determined by Trypan Blue dye exclusion. The ability of these hepatocytes to catalyze certain metabolic transformations such as Phase I and Phase II reactions has been particularly investigated using the benzodiazepine drug, midazolam, as a substance probe. Freshly isolated human hepatocytes in suspension retained the ability to metabolize midazolam to its different hydroxylated derivatives--mainly the 1-hydroxy-midazolam--which was further conjugated with glucuronic acid. For a better understanding of the cytochrome P-450 mediated reactions, we studied the metabolism of midazolam in microsomal fractions prepared from twelve human livers. It was concluded that human microsomes (i) exhibited a Type I binding spectrum upon midazolam addition (Ks = 3.3 microM) and (ii) intensively metabolized the drug to its different derivatives. Furthermore, and since we demonstrated that midazolam was predominantly transformed by a single cytochrome P-450 enzyme, we could attribute the large inter-individual variations in midazolam metabolism to differences in human liver cytochrome P-450 content.

Cytochrome P-450 Enzyme System↗

PubMed1991

Relationships between energy level and insulin secretion in isolated rat islets of Langerhans. A study at various pH values.

To define better the role of [ATP]/[ADP] in insulin release from pancreatic islets, changes in the adenine nucleotide ratios elicited by alterations in external pH were correlated with the secretion profiles produced by administration of two metabolic secretagogues, 16 mM glucose and 10 mM alpha-ketoisocaproic acid. Experiments were carried out in buffers with and without bicarbonate, in the pH range 6.5-7.7. Insulin release was dependent on pHe irrespective of the secretagogue used. Secretion profiles for alpha-ketoisocaproic acid were the same both with and without bicarbonate; the release was decreased below pH 7.1 but maintained at 7.4-7.7. The same pattern was seen with glucose in media buffered with Hepes. With bicarbonate present, secretion caused by high glucose showed a bell-shaped dependence on [H+], with reductions at the acid and alkaline sides of pH 7.1-7.4. [ATP] and [ADP] were higher when Hepes was the buffer, at all pH values studied. The [ATP]/[ADP] declined with increasing pH under both basal and stimulated conditions; the values were always larger after stimulation although at pH 7.7 with bicarbonate present and glucose as the stimulant the difference was very small. It is concluded that: (i) the [ATP]/[ADP] in pancreatic islets is markedly dependent on pHe; (ii) there is no straight-forward correlation between either [ATP] or the absolute value for [ATP]/[ADP] and insulin secretion; and (iii) a rise in [ATP]/[ADP] is necessary for glucose-stimulated insulin release although it is not always the rate-determining event.

Adenosine Diphosphate↗

PubMed1991

Carotid body chemoreception in the absence and presence of CO2-HCO3-.

Carotid body (CB) chemosensory responses to natural and pharmacological stimuli were studied in vitro in the presence and nominal absence of CO2-HCO3- in the perfusion-superfusion media. The CBs obtained from cats (n = 10), anesthetized with sodium pentobarbitone, were simultaneously perfused and superfused with a modified Tyrode solution at 36.5 +/- 0.5 degrees C, equilibrated respectively with PO2 of 120 and less than 20 Torr. The Tyrode, nominally free of CO2-HCO3- (HEPES-NaOH, pH 7.38, 310 mOsm), was used first. Subsequently the Tyrode containing HEPES-HCO3-, equilibrated with PCO2 of 36.8 Torr (pH 7.38) was used. Chemosensory discharges were recorded from the carotid sinus nerve. Both hypoxia (PO2 = 20-25 Torr) and ischemic hypoxia stimulated the discharge in the absence and presence of CO2-HCO3-. However, the presence of CO2-HCO3- significantly raised the baseline activity, augmented the speed, sensitivity and the maximal responses to both types of hypoxia. Hypercapnic perfusate (PCO2 = 65 Torr at pH 7.17) produced a peak response equally promptly in the absence and presence of CO2-HCO3- in the ongoing perfusate but generated a larger and more sustained response. Presence of CO2-HCO3- strongly potentiated the responses to cyanide (10(-10)-10(-7) mol) but less strikingly the responses to nicotine (10(-11)-10(-8) mol). Thus, the extracellular CO2-HCO3- significantly improved the response to hypoxia but was not essential for O2 chemoreception. The underlying mechanisms of the effect of CO2-HCO3- is likely to be mediated by the Cl(-)-HCO3- anion exchanger in the pH regulation of glomus cells.

Animals↗

PubMed1992

K+ and pH homeostasis in the developing rat spinal cord is impaired by early postnatal X-irradiation.

Activity-related transient changes in extracellular K+ concentration ([K+]e) and pH (pHe) were studied by means of ion-selective microelectrodes in neonatal rat spinal cords isolated from pups 2-14 days of age. Pups 1 to 2 days old were X-irradiated to impair gliogenesis and spinal cords were isolated 2-13 days postirradiation (PI). In 2- to 14-day-old pups PI stimulation produced ionic changes that were the same as those in 3- to 6-day-old control (non-irradiated) pups; e.g. the [K+]e increased by 4.03 +/- 0.24 mM (mean +/- S.E.M., n = 30) at a stimulation frequency of 10 Hz and this was accompanied by an alkaline shift of 0.048 +/- 0.004 pH units (mean +/- S.E.M., n = 32) pH units. By contrast, stimulation in non-irradiated 10- to 14-day-old pups produced smaller [K+]e changes, of 1.95 +/- 0.12 mM (mean +/- S.E.M., n = 30), and an acid shift of 0.035 +/- 0.003 pH units which was usually preceded by a scarcely discernible initial alkaline shift, as is also the case in adult rats. Our results show that the decrease in [K+]e ceiling level and the development of the acid shift in pHe are blocked by X-irradiation. Concomitantly, typical continuous development of GFAP-positive reaction was disrupted and densely stained astrocytes in gray matter of 10- to 14-day-old pups PI revealed astrogliosis. In control 3- to 6-day-old pups and in pups PI the stimulation-evoked alkaline, but not the acid, shift was blocked by Mg2+ and picrotoxin (10(-6) M). The acid shift was blocked, and the alkaline shift enhanced, by acetazolamide, Ba2+, amiloride and SITS. Application of GABA evoked an alkaline shift in the pHe baseline which was blocked by picrotoxin and in HEPES-buffered solution. By contrast, the stimulus-evoked alkaline shifts were enhanced in HEPES-buffered solutions. The results suggest a dual mechanism of the stimulus-evoked alkaline shifts. Firstly, the activation of GABA-gated anion (Cl-) channels induces a passive net efflux of bicarbonate, which may lead to a fall in neuronal intracellular pH and to a rise in the pHe. Secondly, bicarbonate independent alkaline shifts may arise from synaptic activity resulting in a flux of acid equivalents.

Animals↗