Aminopyrine Research Articles

Oxidation of pyrazolone pharmaceuticals by peracetic acid: Kinetics, mechanism and genetic toxicity variations

Peracetic acid (PAA) oxidation is an emerging technology in water disinfection and purification. This study evaluated the oxidation of three pyrazolone pharmaceuticals (i.e., Aminopyrine (AMP), Antipyrine (ANT), and Isopropylphenazone (PRP) by PAA. Experimental results showed that PAA exhibited structure selectivity to the above three pharmaceuticals and oxidized AMP with the highest reactivity. The degradation kinetics of AMP was investigated by calculating the apparent second-order rate constants (kapp) under different initial pH. Through kinetic simulation, the second-order rate constants of elementary reactions between AMP (i.e., neutral (AMP0) and protonated (AMP+) species) with PAA (i.e., neutral (PAA0) and anionic (PAA−) species) were obtained to be 0.34 ± 0.077 M−1 s−1(k”AMP+, PAA0), 0.89 ± 0.091 M−1 s−1(k”AMP0, PAA-) and 5.94 ± 0.142 M−1 s−1(k”AMP0, PAA0), respectively. The PAA could oxidize AMP via electrophilic attack, and the degradation site of AMP was confirmed to be the central nitrogen of –N(CH3)2 with the highest relative electrophilicity (sk-/sk+, 48.8614) by Density Functional Theory (DFT) calculation. The intermediates/products of AMP degradation were identified by high-performance liquid chromatography-mass spectrometry (LC-MS/MS), and the transformation pathways of AMP during PAA oxidation were inferred to be hydroxylation, demethylation, and CC cleavage. The genetic toxicity of AMP contaminated water could be reduced after PAA oxidation, which was evaluated by the micronucleus test of Vicia faba root tips.

A Review: Why Aminopyrine Banned?

A Review: Why Aminopyrine Banned?

Degradation behaviors of Isopropylphenazone and Aminopyrine and their genetic toxicity variations during UV/chloramine treatment

Combination of ultraviolet and chloramine (i.e., UV/chloramine) treatment has been attracting increasingly attention in recent years due to its high efficiency in removing trace organic contaminants. This study investigated the degradation behaviors of two pyrazolone pharmaceuticals (i.e., Isopropyl phenazone (PRP) and Aminopyrine (AMP)) and their genetic toxicity variations during UV/chloramine treatment. The results showed that chloramine could hardly degrade PRP and AMP, while UV/chloramine greatly increased the observed first-order rate constant (kobs) of PRP and AMP degradation. The quenching and probe experiments illustrated that the reactive chlorine species (RCS) contributed dominantly to PRP removal, and hydroxyl radical (HO•) was the major contributor to the degradation of AMP, while the reactive amine radicals (RNS) could hardly degrade them. The overall degradation rates of PRP and AMP decreased as pH increased from 6.5 to 10. The kobs of PRP and AMP increased along with NH2Cl dosage increasing and reached a plateau at higher concentrations (0.2–0.5 mM). The present background carbonate (HCO3−, 1–10 mM), chloride (Cl−, 1–10 mM) and natural organic matter (NOM, 5-10 mg-C L−1) exhibited inhibition impacts on PRP and AMP degradation. In addition, the intermediates/products of PRP and AMP were identified and their general degradation pathways were proposed to be hydroxylation, deacetylation, and dephenylization. Specifically, Cl-substitution was inferred during PRP degradation, while demethylation in tertiary amine group was only observed in AMP degradation. These mechanisms including the main reactive sites of PRP and AMP were further confirmed by the frontier orbitals calculation. Moreover, the results of the genetic toxicity according to the micronucleus test of Viciafaba root tip indicated that UV/chloramine treatment could partially reduce the genetic toxicity of PRP and AMP.

Effects of Temperature and Humidity on the Skin Permeation of Hydrophilic and Hydrophobic Drugs.

The humidity was a well-known method to hydrate the skin; however, the published data were varied, and systemic experiments in the previous papers were few. Therefore, the in vitro permeation of excised porcine ear skin by drugs with different polarities [aminopyrine (AMP), antipyrine (ANP), methylparaben (MP), and ibuprofen (IP)] was analyzed under a constant skin surface temperature with different temperatures and humidities to reveal the effects of temperature and humidity on the skin permeation enhancement effects. Applied formulations were prepared by mixing the drug and a hydrophilic vehicle containing glycerin. The disposition-distance profiles of water and the humectant glycerin in the stratum corneum were also investigated using confocal Raman microscopy. High absolute humidity (AH) significantly contributed to the high skin penetration of the hydrophilic penetrants AMP, ANP, and MP but not the hydrophobic penetrant IP. An increase in the partition parameter and a decrease in the diffusivity parameter occurred with an increase in AH, independent of drug polarity. Moreover, we found that dew condensation induced by high AH on temperature-controlled skin surface may effectively increase water content and may provide higher glycerin distribution in the skin barrier, the stratum corneum. Increasing the amount of water and hydrophilic vehicles such as glycerin in the stratum corneum may enhance the permeation of hydrophilic penetrants AMP, ANP, and MP. These data suggested a dew condensation on the skin surface induced by high AH at a constant skin surface temperature would be important to enhance hydrophilic penetrants.

Degradation behaviors and genetic toxicity variations of pyrazolone pharmaceuticals during chlorine dioxide disinfection process

Pyrazolone pharmaceuticals such as isopropylphenazone (PRP) and aminopyrine (AMP) were commonly used as antipyretic analgesics, and their frequently detection in aquatic environment has become a major concern in recent years. Chlorine dioxide (ClO2) disinfection process has been proved to be a feasible way to degrade antipyrine (ANT), which is the first synthetic pyrazolone pharmaceutical. In this work, the reaction kinetics, degradation pathways and genetic toxicity of PRP and AMP during ClO2 disinfection process were investigated. Experimental results demonstrated that the reaction of both PRP and AMP with ClO2 followed second-order kinetics, the second-order rate constants were calculated to be kapp(PRP) = 11.0 M−1 s−1 and kapp(AMP) = 1.30 × 105 M−1 s−1 at neutral pH. Slightly alkaline environment (pH = 9) was in favor of the reaction. CC double bond and CN bond on pyrazolone ring were main active groups under ClO2 electrophilic attack. The degradation pathways of pyrazolone pharmaceuticals could be concluded as CC cleavage, ring-opening reaction and de-carbonyl reaction. Pyrazolone contaminated water exhibited genetic toxicity according to micronucleus test of Viciafaba root tip, however, the toxicity could be reduced through ClO2 disinfection process.

Biodegradation of Methylene Blue Dye by Sequential Treatment Using Anaerobic Hybrid Reactor and Submerged Aerobic Fixed Film Bioreactor

Laboratory scale experiments were carried out to access the feasibility of sequential anaerobic/aerobic biological treatment for the biodegradation of Methylene Blue (MB) dye. Anaerobic studies were performed using anaerobic hybrid reactor (consisting of UASB and Anaerobic filter) whereas submerged aerobic fixed film reactor was used as aerobic reactor. Degradation of MB dye was attempted using neutralized acetic acid (1000 mg/L) as co-substrate. MB dye concentration was stepwise increased from 10 to 70 mg/L after reaching steady state in each dye concentration. Such a gradual increase in the dye concentration helps in the proper acclimatization of the sludge to dyes thereby avoiding the possible inhibitory effects to biological activities at high dye concentrations. The overall treatment efficiency of MB through sequential anaerobic–aerobic reactor operation was 90% at maximum attempted dye concentration of 70 mg/L. The effluent from anaerobic reactor was analysed for intermediate biodegradation products through HPLC. It was observed that catechol, quinone, amino pyrine, 1,4 diamino benzene were present. However they were absent in final effluent.

Transformation of aminopyrine in the presence of free available chlorine: Kinetics, products, and reaction pathways

Aminopyrine (AMP) has been frequently detected in the aquatic environment. In this study, the transformation mechanism of AMP by free available chlorine (FAC) oxidation was investigated. The results showed that FAC reacted with AMP rapidly, and a 74% elimination was achieved for 1.30 μM AMP after 2 min at 14.08 μM FAC dose. AMP chlorination was strongly pH-dependent, and its reaction included second- and third-order kinetic processes. Three active FAC species, including chlorine monoxide (Cl2O), molecular chlorine (Cl2), and hypochlorous acid (HOCl), were observed to contribute to AMP degradation. The intrinsic rate constants of each FAC species with neutral (AMP0) and cation (AMP+) species were obtained by kinetic fitting. Cl2O exhibited the highest reactivity with AMP0 (kAMP0, Cl2O = (4.33 ± 1.4) × 109 M−1s−1). In addition, Cl2 showed high reactivity (106–107 M−1s−1) in the presence of chloride, compared with HOCl (kAMP+, HOCl = (5.73 ± 0.23) × 102 M−1s−1, kAMP0, HOCl = (9.68 ± 0.96) × 102 M−1s−1). At pH 6.15 and 14.08 μM FAC dose without chloride addition, the contribution of Cl2O reached to the maximum (33.3%), but in the whole pH range, HOCl was the main contributor (>66.6%) for AMP degradation. The significance of Cl2 was noticeable in water containing chloride. Moreover, 11 transformation products were identified, and the main transformation pathways included pyrazole ring breakage, hydroxylation, dehydrogenation, and halogenation.

Changes of the toxic potential of drinking water containing aminopyrine before and after chlorine disinfection as determined by the algal toxicity assay and the SOS/umu assay

This paper investigated the potential eco-toxicity changes of the drinking water containing aminopyrine (AMP) before and after chlorination. For the determination of the acute toxicity and genotoxicity, algal toxicity assay and SOS/umu assay were adopted respectively. In these toxicity assays, the AMP exposure group was compared with the control group or the chlorination group to illustrate the changes. Results showed that the algal growth inhibition percentage increased with the increase of AMP concentration. After AMP chlorination, the inhibition percentage decreased at relatively low chlorine ratios (i.e., the AMP to chlorine molar ratio of 4:1 and 1:1) but increased at high chlorine ratios (i.e., the AMP to chlorine molar ratio of 1:2 and 1:5). The superoxide dismutase (SOD) activity was stimulated at AMP to chlorine molar ratios of 4:1 while inhibited at 1:5. The change of malondialdehyde (MDA) content was just opposite to the SOD activity. In SOS/umu assay, when the molar ratios of AMP and chlorine were 1:1, 1:2 and 1:5, the values of induction ratio (IR) were over 1.5 and showed a positive genotoxicity signal in the absence/presence of S9 homogenate. Further, the Toxic Equivalent Quantity (TEQ) value, which was used to express the genotoxic potentials of those positive samples, gradually increased with the increase of chlorine ratio. In a word, the potential genotoxicity of chlorinated drinking water containing AMP should be of a concern.

Aminopyrine Raman spectral features characterised by experimental and theoretical methods: toward rapid SERS detection of synthetic antipyretic–analgesic drug in traditional Chinese medicine

The adulteration of traditional Chinese medicine (TCM) with synthetic drugs severely harms the TCM effect and public health. Thus, quick detection of synthetic drug found in TCM is crucial. Here, we report surface-enhanced Raman scattering (SERS) coupled with density functional theory (DFT) calculations for the direct identification and detection of aminopyrine (AP) in TCM using silver-coated gold nanoparticles (Au@Ag NPs). It has been demonstrated that the Raman enhancement of Au@Ag NPs to AP was stronger than that of Au, Ag and Ag@Au NPs, and there was a strong dependence of the Raman enhancement on the substrate concentration. Moreover, we obtained the DFT calculation result of AP and found that the calculation result was in good agreement with the experimental one. In addition, the characteristic peak at 999 cm−1, which was the strongest peak in the SERS spectra, was used for a quantitative evaluation of the AP level. The limit of detection (LOD) was 2.50 × 10−7 M (equal to 57.8 ng mL−1) in aqueous solution, which was in the same order of magnitude as that obtained by liquid chromatography-mass spectrometry (LC-MS), and the recovery of AP ranged from 93.9% to 101% in the spiked TCM (Shexiang Fengshi capsule) sample. Each test using the SERS sensor only needed a 10 μL sample solution and the whole test could be accomplished within ∼3 min. The SERS technique can be applied to authenticate and detect trace AP in real herbal products.

Coupling Biocatalysis with Molecular Imprinting in a Biomimetic Sensor

Make it simple: A molecularly imprinted electropolymer was combined with an enzyme in a catalytic biomimetic sensor that enabled interference-free detection of the drug aminopyrine (AP) at submicromolar concentrations in the presence of ascorbic acid and uric acid within 15 s. The sensor functioned by the peroxide-dependent conversion of AP in a layer above a product-imprinted electropolymer on an indicator electrode.

An Endogenous Activator Protein of H+, K+‐ATPase Regulates Acid Secretion in Isolated Rabbit Gastric Glands

We previously showed that an endogenous activator protein (HAF) stimulated gastric H+, K+‐ATPase activity in vitro. The present study evaluates the direct role of HAF on intercellular modulation of gastric H+ transport using an HAF‐specific antibody (HAF‐Ab).MethodsCytosolic and membrane fractions of rabbit parietal cells (PC) were analyzed by Western blot using polyclonal HAF‐Ab. The HAF‐Ab was subsequently tested for its effects on H+, K+‐ ATPase activity in PC membrane fractions, and histamine‐stimulated H+ secretion in permeabilized gastric glands (PGG). H+ secretion was measured indirectly using [14C] aminopyrine (AP) uptake ratio.ResultsHAF‐Ab recognized 2 closely apposed bands near the 40 kDa region of the apical and tubulovesicle membranes, and cytosolic fractions. The catalytic α and β subunits of H+, K+‐ ATPase were not recognized by HAF‐Ab. HAF‐Ab dosedependently inhibited HAF‐stimulated membrane H+, K+‐ATPase activity, and decreased AP uptake in histamine‐stimulated PGG. In the latter study, immunofluorescence revealed migration of cytosolic HAF towards the cannelicular plasma membrane, where H+, K+‐ATPase is located.ConclusionsHAF molecules migrate to the secretory plasma membrane following histamine stimulation, and close contact between HAF and H+, K+‐ATPase is essential in the process of gastric acid secretion. HAF‐Ab was found to be specific for HAF, but not for the α and β subunits of H+, K+‐ATPase. Since HAF‐Ab inhibited H+, K+‐ATPase and histamine‐stimulated acid secretion, this suggests, for the first time, evidence for a direct regulatory role of endogenous HAF on gastric H+ transport.

The role of phosphoinositide 3‐kinase (PI3K) in the stimulation of acid secretion by gastric parietal cells

Previous studies using the aminopyrine (AP) assay showed gastric glands treated with histamine (H) + the PI3K inhibitor, LY294002 (H+LY), had enhanced acid secretory response compared to glands stimulated by H alone, and nearly to the extent of maximum response produced by histamine + IBMX (H+I). AP uptakes were 3.5, 8, and 7-fold over resting for H, H+I, and H+LY, respectively. Our data further showed that LY is not acting on PI3K via the Ca2+/PKC pathway or the EGF pathway, and activation of G protein-coupled H2 receptor (H2R) prior to adenylate cyclase is required. We now report on studies to confirm the enhanced secretion of H+LY. Morphology of parietal cells was examined by DIC microscopy and expansion of canalicular area was used to index acid secretion. The increase in area was larger for H+LY than for H alone. Ultrastructural detail of stimulated cells was obtained by electron microscopy using quantitative stereology. For glands stimulated by H+LY microvilli and canalicular volume increased by 4 and 3-fold respectively and tubulovesicle volume decreased by 3-fold compared to resting, substantiating a major morphological response. Relaxation of H+LY stimulated glands by adding the H2R antagonist (lupitidine) was tested. Secretion in response to H+LY slowed after lupitidine suggesting enhanced stimulation by LY was not due to pumps remaining at the apical membrane, but cells were stimulated to a higher degree. Thus, H+LY stimulates the full acid secretory response to a higher degree than H alone, and similarities between H+LY and H+I stimulation suggest this is due to an increase in [cAMP]. We propose PI3K is activated during parietal cell stimulation via H2R and that PI3K modulates an inhibitory pathway that is overwhelmed by increased [cAMP]. Support by DK10141.

Application of Eudragit RS to thermo-sensitive drug delivery systems: II. Effect of temperature on drug permeability through membrane consisting of Eudragit RS/PEG 400 blend polymers

The Eudragit RS and polyethylene glycol 400 (PEG 400) blend polymer (EPG) membranes were prepared by the solvent casting method to pioneer a novel application of Eudragit RS to a thermo-sensitive material. The EPG membranes containing 2.5–10% PEG 400 (2.5–10% EPG) showed the glass transition temperatures (Tgs) around the body temperature (32–42 °C). Drug permeation studies through the EPG membranes were carried out using acetaminophen (AAP) and aminopyrine (AMP) as the model drugs. The permeability of AAP and AMP through the EPG membranes has been shown to be a discontinuous function of temperature, that is, their permeability increased steeply above the Tg of the membranes. The amount of AMP permeated at 42 °C was nearly eight times as much as that at 36 °C. Arrhenius plots of the steady-state permeability coefficient ( P) of AAP indicated two straight lines that intersect at the Tg of the 10% EPG membrane. In the water uptake study for the 10% EPG membrane, the degree of the swelling for the membrane tended to increase with increasing temperature above the Tg of the membrane. The thermo-sensitive permeation mechanism for the EPG membranes might be based on the structure change of the membranes caused by the glass transition, so that the membranes could absorb more water. Considering the high biological safety of Eudragit RS and PEG 400, the EPG membranes might be used to develop a novel thermo-sensitive drug delivery system.

Effects of antioxidant 1-O-hexyl-2,3,5-trimethylhydroquinone or ascorbic acid on carcinogenesis induced by administration of aminopyrine and sodium nitrite in a rat multi-organ carcinogenesis model.

The effect of antioxidant, 0.25% 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ) or 0.25% ascorbic acid (AsA), on Carcinogenesis induced by administration of 0.05% aminopyrine (AP) and 0.05% sodium nitrite (NaNO2), was examined using a rat multi‐organ carcinogenesis model. Groups of twenty F344 male rats were treated sequentially with an initiation regimen of N‐diethylnitrosamine, N‐methyl‐N‐nitrosourea, N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine, N, N′‐dimethylhydrazine and 2,2′‐dihydroxy‐di‐n‐propylnitrosamine during the first 4 weeks, followed by AP+NaNO2, AP+NaNO2+HTHQ, AP+NaNO2+AsA, NaNO2+HTHQ, NaNO2+AsA, each of the individual chemicals alone or basal diet and tap water as a control. All surviving animals were killed at week 28, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. In the AP+NaNO2 group, the incidences of hepatocelluar adenomas and hemangiosarcomas were 95% and 35%, respectively. When HTHQ or AsA was simultaneously administered, the incidences decreased to 58% and 11%, or to 80% and 15%, respectively. On the other hand, in the AP+NaNO2 group and the NaNO2‐alone group, when HTHQ, but not AsA, was simultaneously administered, the incidence of carcinomas in the forestomach significantly increased. The results suggest that HTHQ can prevent tumor production induced by AP and NaNO2 more effectively than AsA. On the other hand, an enhancing or possible carcinogenic effect of simultaneous administration of HTHQ and NaNO2 only on the forestomach is suggested, while simultaneous treatment with the same dose of AsA and NaNO2 may not be carcinogenic to the fore‐stomach or other organs.

An improved method to evaluate secretory activity of isolated gastric glands and cells.

The accumulation of [14C]aminopyrine (AP) is a valuable and widely used method to probe acid secretion of gastric glands and parietal cells. Usually, the dry weight of glands is used to normalize the AP accumulation ratio, and since the nonhomogeneity of the suspension makes it impossible to evenly distribute glands by simple pipetting, it is necessary to scrupulously dry and weigh each and every experimental sample. Thus, massive, time-consuming procedures of tube drying and weighing are involved. Moreover, the weighing of approximately 1 mg dried gland samples in a 1-g Eppendorf tube introduces considerable sample variance. Here, we present a modified protocol to simplify the AP accumulation method by introducing a generic 3H labeling of protein for normalization. Freshly isolated glands were treated with high specific activity 3H-labeled succinimidyl propionate (3H-succ, 60 Ci/mmol) for 10 min at room temperature during the normal isolation/washing procedure. This reagent reacts with primary amines, and even at normal cell pH the efficiency of reaction (25-30%) is more than adequate. The 3H-labeled glands are then processed normally with simultaneous monitoring of 3H (representing gland amount) and AP (representing the extent of acid accumulation) in separate energy windows of a liquid scintillation counter. Dose- and time-dependent efficiency of 3H labeling were evaluated. The relations between labeling and gland protein and dry weight were linear. No detrimental effects of reagent were noted in the useful range of 1-3 nM 3H-succ. Although some limited sample weighing or protein determination must be made for each batch of 3H-labeled glands, this method avoids massive tube weighings and provides the convenience of double label counting with a highly reproducible method for normalizing data.

Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model.

Gastric gland stimulation triggers H(+),K(+)-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLO-permeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H(+),K(+)-ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H(+),K(+)-ATPase-containing tubulovesicles to apical plasma membrane.

Excretory/secretory products of Haemonchus contortus inhibit aminopyrine accumulation by rabbit gastric glands in vitro

Abomasal parasites inhibit gastric acid secretion and reduce the number of acid secreting parietal cells either through physical contact with gastric tissue, the release of inhibitory excretory/secretory (ES) products or by initiating the host inflammatory response. To examine the role of parasite ES products, adult Haemonchus contortus were incubated in a medium based on Hank’s balanced salt solution and three incubates were tested for the presence of inhibitors of acid secretion by dispersed rabbit gastric glands in vitro, using the intracellular accumulation of 14 C -aminopyrine (AP) as an indirect measure of acid secretion. Acceptably sensitive rabbit preparations (80%) for testing ES products showed increased 14 C -AP accumulation with either 10 −5 or 10 −4 M histamine. Sheep glands proved unsuitable as a test system as only two of six preparations responded weakly to histamine. Inhibitory activity was demonstrated in all the three parasite incubates, although responses varied quantitatively between tests, even with the same batch of incubate. In single tests, 16% ES products significantly reduced the 14 C -AP ratio ( P<0.05) of 10 −4 M histamine-stimulated glands (15 of 19 tests with Batch 1 and two of six with Batch 3). Even at 1 and 5%, ES products (Batch 2) were inhibitory for 10 −5 M histamine-stimulated glands: at 1% the mean reduction was 26.0% (range 7.8–54.8%, n=10), four of which were statistically significant and at 5% was 17.6% (range −15.3–53.8%, n=17), four of which were statistically significant. ES products were overall inhibitory (−12%, P<0.05), when tested on glands exposed to increasing histamine concentrations from 10 −6 M to 10 –3 M (which increased the 14 C -AP ratio ( P<0.001)). Responses by individual gland preparations differed ( P<0.001). The active component(s) of the parasite ES products were less than 5000 molecular weight. Ammonium chloride, tested over the range of concentrations of ammonia present in worm incubates (0.2–1 mM, final concentrations in the gland incubations of 0.005–0.1 mM), had variable, but overall inhibitory effects on 10 −5 M histamine-stimulated glands ( n=3). When tested with a range of histamine concentrations, 0.01 mM NH 4Cl overall reduced the response by 8.6% (P<0.05) (n=4) . The similarity of responses of rabbit glands to parasite ES products and to NH 4Cl suggests that ammonia may be the small molecular weight ES product of adult H. contortus, which inhibited acid secretion in vitro.

Comparison of five antisecretory agents acting via gastric H +/K +-ATPase

Lansoprazole(L), pantoprazole (P), rabeprazole ® and RO-18-5364 (RO) are new benzimidazole derivatives which rival omeprazole (O) as proton pump inhibitors (PPIs) for treatment of ulcer disease. In this study, we compared the effects of these compounds on acid secretion and determined their relative potencies in relation to their effect on [ 14C]-aminopyrine (AP) accumulation in isolated gastric glands. Inhibition of AP (1.2 μCi.mL –1) accumulation was measured in rabbit isolated gastric glands. dbcAMP (1 mmol; stimulant of acid secretion) and Ro 20-1724 (0.1 mmol; a phosphodiasterase inhibitor) were added to the Eppendorf tubes containing the PPIs and AP and dose-response curves were done for each drug after incubating for 5, 10 and 20 min at 37 °C and AP accumulation was determined using a scintillation counter. All the PPIs significantly ( P < 0.001) inhibited acid secretion as demonstrated by the inhibition of AP accumulation in the isolated gastric glands. Minimum inhibition occurred at a concentration of 0.001 μmol for lansoprazole and omeprazole, 0.01 μmol for rabeprazole and RO 18-5364 and 0.02 μmol for pantoprazole. No differences were observed between PPIs with regards to the maximum inhibition they produce. When expressed as a percentage inhibition of control at 10-min incubation and at concentrations of 1 μmol, L showed 85.6 ± 0.5, O 87 ± 0.5, P 83.2 ± 1.1, R 86.4 ± 1.1 and RO 87.8 ± 1.9 inhibition respectively. When comparing the IC 50 values, their relative potencies were different. Maximum potency was shown by L (0.007 μmol) > O (0.012 μmol) > R (0.018 μmol) > RO (0.034 μmol) > P (0.050 μmol). All the new PPIs showed different potencies as inhibitors of acid secretion as evident from their IC 50s. Extensive ulcer healing trials demonstrated comparable efficacy with a number of studies indicating that symptoms relief are more rapid with P and L, while in this study L appeared to be the most potent in inhibiting AP accumulation in the isolated gastric glands.

Regulation and function of p38 protein kinase in isolated canine gastric parietal cells.

We examined the regulation and functional role of p38 kinase in gastric acid secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was quantitated by in vitro kinase assay. Carbachol effects were dose- and time-dependent, with a maximal 10-fold stimulatory effect detected after 30 min of incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocked carbachol induction of p38 kinase activity, with maximal inhibition at 10 microM. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca(2+) chelator BAPTA-AM, but incubation of cells in Ca(2+)-free medium led to a 50% inhibition of carbachol induction of p38 kinase activity. Because some of the effects of carbachol are mediated by the small GTP-binding protein Rho, we examined the role of Rho in carbachol induction of p38 kinase activity. We tested the effect of exoenzyme C3 from Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specifically inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inhibited carbachol induction of p38 kinase by 50%. We then tested the effect of SB-203580 and C3 on carbachol-stimulated uptake of [(14)C]aminopyrine (AP). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase in AP uptake induced by carbachol, with maximal (threefold) effect at 10 microM SB-203580. Similarly, preincubation of parietal cells with C3 led to a twofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinase through signaling pathways that are at least in part dependent on Rho activation and on the presence of extracellular Ca(2+). p38 kinase appears to inhibit gastric acid secretion.

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase.

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 +/- 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 +/- 0. 9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 +/- 3.1 and 9.6 +/- 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.