Importance of pretreatment viral load and monitoring of serum hepatitis C virus RNA in predicting responses to interferon-alpha2a treatment of chronic hepatitis C. Hanshin Chronic Hepatitis C Study Group

In order to investigate the conformational structure of isolated human gastric mucus gel pretreated with or without ethanol, gel samples were determined by attenuated total reflection/Fourier transform-infra-red (ATR/FT-IR) microspectroscopy. The result indicates that the secondary structure-dependent amide I band and the glycoprotein region were significantly different in the gastric mucus gels pretreated with and without 40% ethanol. The structural composition of beta-sheet structure (1640-1600 cm-1) increased from 38.48 to 55.08% (+16.6%) after 6-hour pretreatment with 40% ethanol, but the beta-turn structure, (1660-1700 cm-1) decreased from 41.38 to 24.29% (-17.05%). The peak area ranging from 1180 to 1000 cm-1, assigned to the glycoprotein region, was also different after pretreatment with ethanol for 6 h. The higher peak area of the carbohydrate band was obtained in the frequency region between 1000 and 1040 cm-1 and 1100 and 1180 cm-1 for mucus gel pretreated with 40% ethanol. However, the peak area ranging from 1100 to 1040 cm-1 mainly due to the symmetric phosphate stretching mode of proteins was somewhat lower for the ethanol-pretreated mucus gel than the native mucus gel. This result strongly reveals that ethanol significantly modified the conformational structure of proteins and carbohydrates of gastric mucus gel. We propose that the dehydration and interference of hydrophobic interactions in the isolated mucus gel after pretreatment with ethanol might be responsible for this conformational change.     

Laser Raman investigation of the conformation of human immunoglobulin G

Laser Raman spectra of human immunoglobulin G in neutral solution, as well as in the lyophilized and alkaline-denatured states are presented. In the spectrum of the native protein, the amide III band appears at 1240 cm-1 and is assigned to the presence of beta-sheet structure. From its intensity, using a procedure described in this paper, we evaluate the beta-structure content to 37 +/- 4%. This result is supported by the strong amide I' band at 1667 cm-1 and by the presence in the spectra of two bands at 991 and 1078 cm-1, respectively assigned to the C-C and C-N skeletal stretching modes. The differences between the spectrum of the lyophilized powder and that of the solution show that the lyophilization process induces conformational changes that perturb the local environment of some of the tryptophan residues and alter the secondary structure of immunoglobulin G. The beta-structure appears to be more uniform and more abundant in solution. When the protein is denatured at pH 11, the amide III and amide I'bands, which become weaker and broader, shift in frequency from 1240 to 1248 cm-1 and from 1667 to 1656 cm-1 respectively. These changes indicate a decrease in the amount of beta-structure and a transition toward a much more disordered conformation. During the denaturation, the intensities of many bands of the aromatic chromophores change, notably the tryptophan peaks at 879, 1359 and 1573 cm-1.     

Development of an anti-bleeding agent for recombinant hirudin induced skin bleeding in the pig

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.     

Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study

The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.     

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% alpha-helix (amide I and III components at 1653 cm-1, and 1312 and 1284 cm-1, respectively), 40% beta-sheets (amide I and III components at 1625 and 1231 cm-1, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm-1, and 1249 cm-1, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin beta-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (alpha-helix, beta-sheets, and turns) are accurate.     

Neurotrophic effects of BDNF and CNTF, alone and in combination, on postnatal day 5 rat acoustic ganglion neurons

The effects of encapsulating bovine hemoglobin (BHb) in the bicontinuous cubic phase formed by monooleoylglycerol and water was investigated with Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction. Cubic phase was formed in the presence of 1-10 wt% BHb. Studies using X-ray diffraction reveal that at 0.5-2.5 wt% BHb, the cubic phase structure is characterized by the double diamond lattice (Pn3m). At 2.5-5 wt% BHb, coexistence of two cubic phase structures, Pn3m and the gyroid lattice (Ia3d), was observed while at BHb, concentrations higher than 5 wt% the gyroid structure persists. FTIR shows there is an increase in intensity of the free nu C = O (1745 cm-1) and a corresponding decrease in the intensity of the hydrogen bonded nu C = O (1720 cm-1) as the BHb concentration is increased. The nu C-O-CO peak shifts from 1183 cm-1 to 1181 cm-1 as the concentration of BHb raised from 2.5 to 10 wt% indicating BHb may induce subtle changes in the interfacial region of cubic phase monoolein. The bandwidth of the nu asCH2 stretch (2926 cm-1) increased in the presence of 5 wt% BHb compared to samples with 2.5 or 10 wt% BHb. The increase in frequency of the nu sCH2 stretch (2854 cm-1) induced by increasing temperature 20 to 60 degrees C was dampened when BHb was present compared to samples heated in isotonic buffer. Analysis of the amide I band at 1650 cm-1 showed that the secondary structure of BHb is not affected by encapsulation in monoolein. In vitro release studies showed that 45% of the entrapped BHb was released after 144 h at 37 degrees C. The porous nature of bulk cubic phase was further demonstrated by diffusion of K2Fe(CN)6 and conversion of 73% of the oxyhemoglobin to methemoglobin after 1 h. These results suggest that the cubic phase may be useful for encapsulation of Hb as a red cell substitute and for the encapsulation and delivery of other bioactive agents.     

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Strain-dependent differences in beta-sheet conformations of abnormal prion protein

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.     

Correction of complete transposition of great vessels by the Mustard technic (26 cases operated)

Laser Raman spectra of neurotoxins of Pelamis platurus (yellow-bellied sea snake) and Laticauda semifasciata (broad-banded blue sea snake) were investigated. The amide I band appeared at 1672 cm-1 for both toxins, which presents an indication of anti-parallel beta structure. Since this agrees well with the result from the CD-ORD studies of snake neurotoxin, it was concluded that snake neurotoxins mainly consist of beta structure. The amide III band appeared at 1245 cm-1 for P. platurus toxin and 1248 cm-1 for L. semifasciata toxin. The four disulfide bonds present in the toxin have a very similar geometry. After vigorous heat treatment, the backbone configuration of the toxin molecule basically remained the same although it was partially denatured. The major peak at 512 cm-1 was not altered by the heat treatment but a new shoulder appeared at 546 cm-1. This suggests that a new type of S-S stretching vibration (trans-gauche-trans) was produced as a result of heat treatment. However, the majority of the S-S vibrations remained in the gauche-gauche-gauche orientation. A substantial change in the interactions between a tyrosine aromatic ring and neighboring residues was apparently the alteration caused by the heat treatment.     

Effect of deuteration on some IR-spectral characteristics of gelatin

The IR-spectra of normal and deuterated gelatin samples were studied. The 3300 cm-1 band is determined by the valence vibrations of the peptid bond NH-groups, OH-groups of oxyproline and structural water. The 1280-1220 cm-1 bands cannot be intepreted for gelatin as amide III; their appearance is caused by the skeleton vibrations. The 1460 cm-1 band is not Amide II in gelatin, it is associated with the deformation vibrations in free methyl groups of the amino acid residues. The effect of OH-groups of hydration water forming the intramolecular hydrogen bond is displayed by 1670 cm-1 band. Disappearance of the 1560 and 1530 cm-1 bands with deuterating and appearance of the 1580 cm-1 band may evidence for a structural transition of the gelatin molecule from one conformation to another, is more ordered, conformation.     

Involvement of free radicals and histamine in the potentiating action of cigarette smoke exposure on ethanol-induced gastric mucosal damage in rats

Cigarette smoking has been associated with peptic ulcer diseases. We studied the effects of cigarette smoke exposure on ethanol-induced gastric mucosal damage and its relationship with vascular integrity and the possible role of free radicals and histamine. Male Sprague-Dawley rats were exposed to cigarette smoke followed by ethanol administration (70% v/v). Smoke exposure alone dose-dependently reduced basal blood flow and increased xanthine oxidase (XO) activity but superoxide dismutase (SOD) activity remained unaffected in gastric mucosa. Cigarette smoking followed by ethanol administration significantly potentiated mucosal lesion formation along with augmentation of the mucosal blood flow, vascular permeability and myeloperoxidase (MPO) activity. The potentiating effect of smoking on ethanol-induced gastric mucosal lesion and MPO activity was abolished by pretreatment with allopurinol, terfenadine or ranitidine. Terfenadine and ranitidine also reduced the increased mucosal blood flow and vascular permeability induced by smoking and ethanol combined. These findings suggested that cigarette smoke adversely affected the defense mechanisms of the gastric mucosa by reducing the mucosal blood flow which in turn led to ischemia and increased XO activity. Activation of XO together with histamine H1 and H2 receptors stimulation could lead to neutrophil aggregation and vascular damage. However, the potentiating action of cigarette smoke on ethanol ulceration is unlikely through reduction of SOD activity in gastric mucosa.     

Conformational changes in the core structure of bacteriorhodopsin

Bacteriorhodopsin (bR) is the light-driven proton pump found in the purple membrane of Halobacterium salinarium. In this work, structural changes occurring during the bR photocycle in the core structure of bR, which is normally inaccessible to hydrogen/deuterium (H/D) exchange, have been probed. FTIR difference bands due to vibrations of peptide groups in the core region of bR have been assigned by reconstituting and regenerating delipidated bR in the presence of D2O. Exposure of bR to D2O even after long periods causes only a partial shift of the amide II band due to peptide NH --> ND exchange only of peripheral peptide structure. However, the amide II band completely downshifts when reconstitution/regeneration of bR is performed in the presence of D2O, indicating that almost the entire core backbone structure of bR undergoes H/D exchange. Peripheral regions can then be reexchanged in H2O, leaving the core backbone region deuterated. Low-temperature FTIR difference spectra on these core-deuterated samples reveal that peptide groups in the core region respond to retinal isomerization as early as the K intermediate. By formation of the M intermediate, infrared differences in the amide I region are dominated by much larger structural changes occurring in the core structure. In the amide II region, difference bands appear upon K formation and increase upon M formation which are similar to those observed upon the cooling of bacteriorhodopsin. This work shows that retinal isomerization induces conformational changes in the bacteriorhodopsin core structure during the early photocycle which may involve an increase in the strength of intramolecular alpha-helical hydrogen bonds.     

A correlative study on the mechanism of adaptive cytoprotection against ethanol-induced gastric lesion formation in rats

The protective effect of mild irritants against the subsequent gastric injury induced by necrotizing agents has been termed 'adaptive cytoprotection'. In this study, the possible pathway and mechanisms of adaptive cytoprotection induced by 20% ethanol were investigated. An ex-vivo gastric chamber preparation was used. The gastric mucosa was exposed to 20% ethanol before subsequent administration of 100% ethanol 15 min later. Subdiaphragmatic vagotomy or drug pretreatment was carried out in order to elucidate the mechanisms of adaptive cytoprotection by 20% ethanol. The results showed that 20% ethanol pre-exposure significantly protected the gastric mucosa against damage caused by 100% ethanol. This protective action was completely abolished by atropine or lidocaine pretreatment, whereas vagotomy and hexamethonium failed to have a significant influence. The cytoprotective effect, however, was independent of the gastric secretory volume, titratable acid content, luminal soluble mucus level and gastric mucosal blood flow. Exposure of only half the gastric mucosa to the mild irritant resulted in the protection of both sides of the mucosa. All these findings indicate that the adaptive cytoprotection of 20% ethanol involves the participation of chemoreceptors and muscarinic receptors in the gastric mucosa. An internal enteric reflex arc, with transmission of signals within the gastric mucosa, may also contribute to the cytoprotective process of the mild irritant.     

Involvement of glutamic acid 278 in the redox reaction of the cytochrome c oxidase from Paracoccus denitrificans investigated by FTIR spectroscopy

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and M?ntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.     

Binding of transducin and transducin-derived peptides to rhodopsin studies by attenuated total reflection-Fourier transform infrared difference spectroscopy

Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent absorption increases occur at 1735 cm-1, 1640 cm-1, 1550 cm-1, and 1517 cm-1. The D2O sensitivity of the broad carbonyl stretching band around 1735 cm-1 indicates that a carboxylic acid group becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.     

Conformational changes in bacteriorhodopsin associated with protein-protein interactions: a functional alpha I-alpha II helix switch?

Fourier transform infrared spectra of bacteriorhodopsin samples were obtained in conditions in which the aggregation state of the protein (i.e., monomeric or trimeric) was modified by different treatments. Two approaches were followed: (1) renaturation of bacteriorhodopsin starting from bacterioopsin dissolved in SDS and (2) reconstitution of bacterioopsin in Halobacterium lipid liposomes at two different lipid/protein ratios. Concomitant with the gradual recovery of the native interactions between transmembrane helices, we observed clear and gradual changes in the infrared absorption spectra in the amide I band and also in the band at 1741 cm-1. These processes were found to be compatible with the two-state oligomerization model. The whole set of experiments shows that the band at 1665 cm-1 in the deconvoluted spectra appears only when monomers interact forming trimers, even when the lattice is not present. This implies that the trimeric organization of bacteriorhodopsin is responsible for the unique features described in the amide I of purple membrane. The spectroscopic changes detected can be attributed to changes in secondary structure compatible with the interconversion of alpha I and alpha II helices. However, the exact nature and functional relevance of these changes is still unknown.     

Histological study of mechanisms of adaptive cytoprotection on ethanol-induced mucosal damage in rat stomachs

Adaptive cytoprotection in the gastric mucosa could be induced by exposure to low concentrations of noxious agents. However, experimental results reported so far were based on macroscopic studies. We aimed to investigate the phenomenon of gastric adaptive cytoprotection of mild irritants and its correlation with intramucosal mucus at the histological level. It was found that histological damage induced by ethanol had a negative correlation with the length of the mucus-secreting layer in the gastric mucosa. Mild irritants such as 20% ethanol and 5% NaCl preserved the 100% ethanol-induced intramucosal mucus depletion, but only the former agent demonstrated a cytoprotective effect against the histological damage, indicating that preservation of intramucosal mucus may not necessarily play a permissive role in adaptive cytoprotection. The capsaicin-sensitive sensory afferent neurons, sensory chemoreceptors, muscarinic receptors, alpha2-adrenoceptors and peripheral dopamine D2-receptors were found to be the components of the autonomic nervous system involved in the cytoprotective processes of 20% ethanol. Endogenous mediators including nitric oxide, prostaglandins, and possibly nonprotein sulfhydryl compounds also seemed to participate in such protection. Nevertheless, 0.3 M HCl did not show any effect either on mucosal damage or depletion of intramucosal mucus induced by absolute ethanol. These findings suggest that only 20% ethanol shows histological cytoprotection, which would involve various components of the autonomic nervous system and endogenous mediators. Furthermore, this investigation also implies a new perspective: that in order to study a true adaptive cytoprotection, histological examination of the gastric mucosa should be performed.     

The effect of dopamine and PGI2 on indomethacin and alcohol-induced gastric lesions in rats of various ages in relation to gastric vascular permeability and HCl secretion

Gastric lesions induced by indomethacin (20 mg.kg-1 i.p.) and ethanol (1 ml 95% intragastrically) were studied in rats after a 24 hour fast. The size of gastric lesions was correlated with gastric HCl secretion and with gastric vascular permeability (determined from the Evan's blue concentration in the gastric tissue after its i.v. administration). These parameters were also studied in rats pretreated with either PGI2 (5 micrograms.kg-1) or dopamine (0.5 mg.kg-1). It was found that in 12-months old rats the gastric lesions were significantly higher compared with the 3-month old group. PGI2 and dopamine significantly decreased gastric lesions in the 3-month rats but not in 12-month old rats. Both indomethacin and ethanol increased gastric vascular permeability in both age groups. It was observed that the decrease of gastric lesions after pretreatment with PGI2 and dopamine in the 3-month old rats was followed with decreased gastric vascular permeability and HCl secretion. On the other hand the increased susceptibility of the gastric mucosa due to indomethacin in the 12-month old rats was followed by a decrease of HCl secretion. PGI2 or dopamine had any effect on the 12-month old rats. These results show that susceptibility of gastric mucosa to PGI2 and dopamine is dependent on age. (Fig. 3, Ref. 31.)     

A dual effect of 1-methyl-4-phenylpyridinium (MPP+)-analogs on the respiratory chain of bovine heart mitochondria

Various gastrointestinal functions such as mucosal blood flow and mucus secretion can be influenced immunologically. Rats were systemically sensitized with 4-hydroxy-3-iodo-5-nitro-phenylacetic acid (NIP), a synthetic antigen. Mucosal release of gastrin, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and leukotriene C4 was measured after intragastric or in vitro antigen challenge. Gastric protection from ethanol was determined. In sensitized rats, intragastric antigen challenge increased release of gastrin from the antral mucosa ex vivo and tended to increase release of prostaglandin F2 alpha. Likewise, antral mucosa of sensitized rats released significantly more gastrin and prostaglandin F2 alpha during in vitro antigen challenge than during incubation in the absence of antigen. Release of 6-keto-prostaglandin F1 alpha and leukotriene C4 was not affected by the immunologic reaction. Topical antigen challenge in sensitized rats reduced gastric mucosal damage caused by ethanol by 50%. The immunologically induced gastroprotection was significantly attenuated by pretreatment with indomethacin. The findings show that specific antigen challenge renders the gastric mucosa more resistant against the injurious effect of ethanol indicating that the stomach is a target organ of immunological reactions. As gastrin and prostaglandins exert potent protective effects, release of these mediators may contribute to the protective response to gastric mucosal immune activation.