Bioimpedance spectroscopy: a new tool to assess early esophageal changes linked to gastroesophageal reflux disease?

Bioimpedance spectroscopy can identify pathological changes related to precancerous lesions of the cervix uteri and esophagus. It therefore has the potential to detect early reflux-related changes in the esophageal mucosa, such as dilated intercellular spaces. The reliable detection of dilated intercellular spaces at the time of endoscopy would yield a significant diagnostic advantage for separating patients with functional heartburn from the large proportion of patients with gastroesophageal reflux symptoms but no macroscopic esophagitis or pathological acid exposure. The bioimpedance of the esophageal mucosa, measured with a small caliber probe, was evaluated in a series of preclinical experiments. First, sections of rabbit esophageal epithelium were mounted in Ussing chambers and exposed to solutions at pH 7.4 or pH 1.5 for 45 minutes. Impedance measurements were taken at varying probe pressures. Second, rabbit esophageal epithelia were perfused for 45 minutes in situ with pH 1.1 or control solutions and impedance measurements taken. Samples from both in vitro and in situ experiments were taken for morphological examination by light microscopy. Finally, esophageal bioimpedance was measured in awake dogs with permanent esophagocutaneous stoma. The in situ experiments demonstrated that morphological changes in the esophageal mucosa could be discerned by the use of bioimpedance spectroscopy. The variability in resistivity was species-independent but was affected by the pressure applied to the probe. The results suggest that evaluation of bioimpedance spectroscopy for use in a clinical setting is warranted. Small morphological differences in the esophageal mucosa may be detected by the use of bioimpedance spectroscopy.

Gastrointestinal-specific anxiety: an important factor for severity of GI symptoms and quality of life in IBS.

BACKGROUND: Gastrointestinal (GI)-specific anxiety (GSA) has been proposed to influence symptom severity and quality of life (QOL) in patients with irritable bowel syndrome (IBS). The Visceral Sensitivity Index (VSI) is a recently developed, reliable and valid measure of GSA. Our aim was to evaluate the association between GSA, GI symptom severity, and QOL in IBS patients. METHODS: Sixty healthy subjects and 306 patients fulfilling the Rome II criteria for IBS were studied. Demographic and disease-related factors were assessed. Patients completed VSI and GI Symptom Rating Scale (GSRS) and questionnaires to determine psychological symptom severity (Hospital Anxiety and Depression Scale), QOL (Short form 36), and presence of functional GI disorders (Rome II Modular Questionnaire). KEY RESULTS: Compared with healthy subjects, patients with IBS had more severe GSA (34.7 +/- 16.9 vs. 2.2 +/- 4.4 [mean +/- standard deviation]; P < 0.0001). In the IBS group, more severe GSA was seen in patients with more severe GI symptoms (P < 0.0001), general anxiety (P < 0.0001) and depression (P < 0.0001), and with lower socioeconomic status (P < 0.05). In a regression analysis, GSA was the strongest predictor for GI symptom severity (GSRS total score), followed by number of Rome II diagnoses, presence of meal-related IBS symptoms, and gender (R(2) = 0.34). Gastrointestinal-specific anxiety was also, together with general anxiety, depression, socioeconomic status, and gender, found to be independently associated with mental QOL (R(2) = 0.62). CONCLUSIONS & INFERENCES: Gastrointestinal-specific anxiety seems to be an important factor for GI symptom severity and QOL in patients with IBS.

A new intelligent distension system for hollow organs.

Controlled distension of hollow organs is an accepted technique for generating reproducible visceral stimuli. We have constructed a new, flexible and intelligent distension system in which discomfort, pain and autonomic responses are recorded online. These responses can be fed back into the system in a regulatory loop and be used to shape the distension paradigm. Consequently, it is possible to take all subjects to a state of equal, although subjective, level of discomfort or pain, even though pressure, tension and volume might be totally different. By using a variable airflow, this new distension system can be effectively used in all kinds of paradigms, e.g. phasic, tonic, or ramp distensions or customized combinations of them. The system can be used to control pressure, volume or tension. A refinement of the system is that it is possible to automatically change the controlled entity during a distension, e.g. from an isobaric ramp directly into an isovolumetric tonic phase. Furthermore, the distension device allows double distensions with independent distension paradigms.

Formation of the "peroxy" intermediate in cytochrome c oxidase is associated with internal proton/hydrogen transfer.

When dioxygen is reduced to water by cytochrome c oxidase a sequence of oxygen intermediates are formed at the reaction site. One of these intermediates is called the "peroxy" (P) intermediate. It can be formed by reacting the two-electron reduced (mixed-valence) cytochrome c oxidase with dioxygen (called P(m)), but it is also formed transiently during the reaction of the fully reduced enzyme with oxygen (called P(r)). In recent years, evidence has accumulated to suggest that the O-O bond is cleaved in the P intermediate and that the heme a(3) iron is in the oxo-ferryl state. In this study, we have investigated the kinetic and thermodynamic parameters for formation of P(m) and P(r), respectively, in the Rhodobacter sphaeroides enzyme. The rate constants and activation energies for the formation of the P(r) and P(m) intermediates were 1.4 x 10(4) s(-1) ( approximately 20 kJ/mol) and 3 x 10(3) s(-1) ( approximately 24 kJ/mol), respectively. The formation rates of both P intermediates were independent of pH in the range 6.5-9, and there was no proton uptake from solution during P formation. Nevertheless, formation of both P(m) and P(r) were slowed by a factor of 1.4-1.9 in D(2)O, which suggests that transfer of an internal proton or hydrogen atom is involved in the rate-limiting step of P formation. We discuss the origin of the difference in the formation rates of the P(m) and P(r) intermediates, the formation mechanisms of P(m)/P(r), and the involvement of these intermediates in proton pumping.

Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase.

We have investigated the electron-proton coupling during the peroxy (P(R)) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of Cu(A) is increased by approximately 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of approximately 200. In ED(I-286) proton uptake during P(R)-->F was slowed by a factor of approximately 5, which indicates that E(I-286) is the proton donor to P(R). In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of approximately 2.5 as compared with approximately 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Adelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of approximately 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.

Localized control of proton transfer through the D-pathway in cytochrome c oxidase: application of the proton-inventory technique.

In the reaction cycle of cytochrome c oxidase from Rhodobacter sphaeroides, one of the steps that are coupled to proton pumping, the oxo-ferryl-to-oxidized transition (F --> O), displays a large kinetic deuterium isotope effect of about 7. In this study we have investigated in detail the dependence of the kinetics of this reaction step ¿k(FO)(chi) on the fraction (chi) D(2)O in the enzyme solution (proton-inventory technique). According to a simplified version of the Gross-Butler equation, from the shape of the graph describing k(FO)(chi)/k(FO)(0), conclusions can be drawn concerning the number of protonatable sites involved in the rate-limiting proton-transfer reaction step. Even though the proton-transfer reaction during the F --> O transition takes place over a distance of at least 30 A and involves a large number of protonatable sites, the proton-inventory analysis displayed a linear dependence, which indicates that the entire deuterium isotope effect of 7 is associated with a single protonatable site. On the basis of experiments with site-directed mutants of cytochrome c oxidase, this localized proton-transfer rate control is proposed to be associated with glutamate (I-286) in the D-pathway. Consequently, the results indicate that proton transfer from the glutamate controls the rate of all events during the F --> O reaction step. The proton-inventory analysis of the overall enzyme turnover reveals a nonlinear plot characteristic of at least two protonatable sites involved in the rate-limiting step in the transition state, which indicates that this step does not involve proton transfer through the same pathway (or through the same mechanism) as during the F --> O transition.

The onset of the deuterium isotope effect in cytochrome c oxidase.

We have investigated the dynamics of proton equilibration within the proton-transfer pathway of cytochrome c oxidase from bovine heart that is used for the transfer of both substrate and pumped protons during reaction of the reduced enzyme with oxygen (D-pathway). The kinetics of the slowest phase in the oxidation of the enzyme (the oxo-ferryl --> oxidized transition, F --> O), which is associated with proton uptake, were studied by monitoring absorbance changes at 445 nm. The rate constant of this transition, which is 800 s(-)(1) in H(2)O (at pH approximately 7.5), displayed a kinetic deuterium isotope effect of approximately 4 (i.e., the rate was approximately 200 s(-)(1) in 100% D(2)O). To investigate the kinetics of the onset of the deuterium isotope effect, fully reduced, solubilized CO-bound cytochrome c oxidase in H(2)O was mixed rapidly at a ratio of 1:5 with a D(2)O buffer saturated with oxygen. After a well-defined time period, CO was flashed off using a short laser flash. The time between mixing and flashing off CO was varied within the range 0. 04-10 s. The results show that for the bovine enzyme, the onset of the deuterium isotope effect takes place within two time windows of O transition is internal proton transfer from a site, proposed to be Glu (I-286) (R. sphaeroides amino acid residue numbering), to the binuclear center. The spontaneous equilibration of protons/deuterons with this site in the interior of the protein is slow (approximately 1 s).

Potentiation and depression following stimulus interruption in young rat hippocampi.

We examined the effect of stimulus interruption on dual component field EPSPs in the hippocampal CA1 region. Resuming test stimulation at 0.1 Hz after 10-60 min silent periods led to an increase of the response followed by a decline, involving AMPA and NMDA components to a similar extent. Similar changes were seen when stimulation was initially applied to a naive pathway or the stimulus strength was increased during an experiment. The potentiation of the AMPA response was largely blocked by prior application of the NMDA antagonist AP5 while application of this drug immediately after the initial potentiation prevented the following decline. The results demonstrate that NMDA-dependent potentiation and depression, possibly equivalent to LTP and LTD, can both be induced by the same, very low, test stimulus frequency. Furthermore, the depression appeared to have a longer time window for its induction than the potentiation.

Proton uptake controls electron transfer in cytochrome c oxidase.

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the "peroxy" to "oxoferryl" transition, internal transfer of the fourth electron from CuA to heme a has the same rate as proton uptake from the bulk solution (8,000 s-1). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a CuA ligand, was replaced by leucine, which resulted in an increased redox potential of CuA. During reaction of the reduced mutant enzyme with O2, a proton was taken up at the same rate as in the wild-type enzyme (8,000 s-1), whereas electron transfer from CuA to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from CuA to heme a were blocked, the results from this study show that the CuA --> heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a3-CuB before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.

Electron-proton interactions in terminal oxidases.

The cytochrome c and ubiquinol oxidases discussed in this article are membrane-bound redox-driven proton pumps which couple an electron current to a proton current across the membrane. This coupling requires a control of the thermodynamics and/or rates of internal electron- and proton-transfer reactions (termed 'gating'). Therefore, to understand the structure-function relation of these proton pumps, individual electron- and proton-transfer reactions must be investigated. We have undertaken such studies by using a combination of site-directed mutagenesis and spectroscopic techniques. The results show that proton uptake/release upon reduction/oxidation of heme a3 takes place on a ms-time scale through the K-pathway (including Thr(I-359) and Lys(I-362)), but not through the D-pathway (including Asp(I-132) and Glu(I-286)). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway (but not through the K-pathway) in a biphasic process with time constants of 100 microseconds and 1 ms. Thus, the original assignment of the role of the D-pathway (used only for pumped protons) must be revised. Dynamic studies of proton uptake to the enzyme surface show that on the proton-input side, the surface carries a proton-collecting antenna made of carboxylate and histidine residues which enable the enzyme to pick up protons with a rate compatible to the enzyme turnover rate. These results are consistent with the three-dimensional cytochrome c oxidase structure which shows that the entry point to the D-pathway (but not to the K-pathway) is surrounded by a network of histidine residues within a negative electrostatic potential.

Photoinduced electron transfer from carboxymethylated cytochrome c to plastocyanin.

Photoinduced electron transfer from cytochrome c to plastocyanin was investigated using a novel method. Reduced carboxymethylated cytochrome c (CmCyt c), with carbon monoxide bound to the heme iron, and oxidized plastocyanin were mixed. At 1 mM CO the reduced state of CmCyt c is stabilized by about 350 meV. After flash photolysis of CO the apparent redox potential of CmCyt c drops resulting in electron transfer to plastocyanin. The electron transfer characteristics were investigated at approximately 30 different wavelengths in the range 390-460 nm. A global fit of the data showed that the electron transfer rate is 960+/-30 s-1 at pH 7.

Electron paramagnetic resonance studies of the soluble CuA protein from the cytochrome ba3 of Thermus thermophilus.

The electron paramagnetic resonance (EPR) spectrum of the binuclear CuA center in the water-soluble subunit II fragment from cytochrome ba3 of Thermus thermophilus was recorded at 3.93, 9.45, and 34.03 GHz, and the EPR parameters were determined by computer simulations. The frequency and M1 dependence of the linewidth was discussed in terms of g strain superimposed on a correlation between the A and g values. The g values were found to be gx = 1.996, gy = 2.011, gz = 2.187, and the two Cu ions contribute nearly equally to the hyperfine structure, with magnitude of Ax magnitude of approximately 15 G, magnitude of Ay magnitude = 29 G, and magnitude of Az magnitude of = 28.5 G (65Cu). Theoretical CNDO/S calculations, based on the x-ray structure of the Paracoccus denitrificans enzyme, yield a singly occupied antibonding orbital in which each Cu is pi*-bonded to one S and sigma*-bonded to the other. In contrast to the equal spin distribution suggested by the EPR simulations, the calculated contributions from the Cu ions differ by a factor of 2. However, only small changes in the ligand geometry are needed to reproduce the experimental results.

The complementary nature of long-term depression and potentiation revealed by dual component excitatory postsynaptic potentials in hippocampal slices from young rats.

Homosynaptic long-term depression and long-term potentiation were studied in hippocampal slices from 12-18-day-old rats using field excitatory postsynaptic potentials recorded in the CA1 subfield (stratum radiatum). Independent estimates of the alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and the N-methyl-D-aspartate receptor-mediated components of the field excitatory postsynaptic potential were obtained in parallel using early and late measurements of a dual component excitatory postsynaptic potential in a solution containing low (0.1 mM) magnesium and 1 microM of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Long-term depression, induced by 2 Hz stimulation for 10 min, was observed as an equal relative depression of the AMPA and N-methyl-D-aspartate receptor-mediated components of the field excitatory postsynaptic potential, whereas long-term potentiation induced by single or repeated high-frequency stimulation, was seen initially as a predominant potentiation of the AMPA receptor-mediated component. Within the first 30-60 min, long-term potentiation gradually changed to more equal increases of the two components of the excitatory postsynaptic potential. During alternating induction of long-term depression and long-term potentiation, the AMPA and N-methyl-D-aspartate receptor-mediated components could both be repeatedly regulated up and down. Long-term depression and long-term potentiation also showed several signs of interaction with each other during such experiments; e.g., long-term depression removed the occlusive effect of large long-term potentiation on a subsequent long-term potentiation, and long-term potentiation applied after the induction of long-term depression was found to be more stable than otherwise. The results support the notion that long-term depression and long-term potentiation employ changes in a common synaptic property. A tentative mechanism for this modification, expressed as equal changes of AMPA and N-methyl-D-aspartate receptor-mediated components of the excitatory postsynaptic potential, is an alteration in transmitter release, while the initial asymmetric part of long-term potentiation indicates involvement of an additional short-term modification.

Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

On the linkage between AMPA and NMDA receptor-mediated EPSPs in homosynaptic long-term depression in the hippocampal CA1 region of young rats.

Homosynaptic long-term depression (LTD) was studied in hippocampal slices from 12-18-d-old rats using field EPSP recording in the apical dendritic layer of CA1 pyramidal cells. Independent estimates of the alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and the N-methyl-D-aspartic acid (NMDA) receptor-mediated components of the field EPSP were obtained in parallel using early and late measurements of a dual-component EPSP in a low-magnesium solution. LTD was induced by low-frequency stimulation (LFS; 2 Hz for 10 min), resulting in equal relative changes of the AMPA and NMDA receptor-mediated components. Under conditions when the AMPA receptor-mediated component was fully blocked, a similarly sized LTD was observed for the pure NMDA receptor-mediated EPSP (measured as initial slope or peak amplitude). Equal changes in AMPA and NMDA receptor-mediated components occurred also upon application of the adenosine agonist N6-cyclohexyladenosine (CHA), known to act by decreasing transmitter release. On the other hand, LTD was found to interact in a multiplicative manner with the presynaptic release changes induced by CHA and by paired-pulse facilitation. The induction of the LTDs of both AMPA and NMDA receptor-mediated EPSPs was blocked by the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid and by the phosphatase inhibitor okadaic acid. Partial blockade of LTD by okadaic acid resulted in equal partial blockade of the LTDs of the AMPA and NMDA receptor-mediated components. On the other hand, the L-type calcium channel blocker nifedipine, the metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine, the nitric oxide synthase inhibitor N omega-nitro-L-arginine, and the heme oxygenase inhibitor protoporphyrin IX zinc(II) had no effect on LTD of either the AMPA or the NMDA receptor-mediated component. These results of equal changes of AMPA and NMDA receptor-mediated components of the field EPSP in association with LTD, and the consistent parallelism of effects or noneffects on these components by various receptor antagonists and enzyme inhibitors, seem more easily explained by a presynaptic locus for LTD than by a postsynaptic one.