血清胃泌素变化与急性胃粘膜病变关系的研究

对大白鼠血清中胃泌素水平的变化与急性胃粘膜病变的关系进行了初步的研究,结果表明以消炎痛为诱因引起的急性胃粘膜病变大白鼠血清胃泌素水平明显增高。而维酶素可以抑制因消炎痛引起的急性胃粘膜病变时血清胃泌素的释放,对胃粘膜具有保护作用。     

胃(H~ K~ )-ATPase结构与功能的研究

胃(H~ K~ )-ATPase属于生物膜的第二类质子泵(E_1E_2型),从生理角度它是胃酸分泌的质子泵。本文结合我们初步的研究结果:猪、大白鼠胃粘膜(H~ K~ )-ATPase的纯化以及由消炎痛引起的急性胃粘膜病变与胃粘膜(H~ K~ )-ATPase的关系等,对此酶在近十几年来它的纯化、结构、性质、催化机理,向胃腔分泌盐酸的功能及其调节和胃病变的分子机理等方面进行了简要的综述。     

消炎痛对猪胃H~ /K~ -ATP酶质子转运功能的抑制

作为猪胃H~ /K~ -ATPase的非竞争性抑制剂,消炎痛明显抑制H~ /K~ -ATPase泡囊的质子转运功能,造成质子泄漏。在0.15 mg/ml蛋白浓度下,4%的消炎痛结合于H~ /K~ -A- TPase泡囊上。它能渗入膜脂相并显著降低膜的流动性,并使H~ /K~ -ATPase内源荧光受到淬灭。从实验结果看来,消炎痛对猪胃H~ /K~ -ATPase质子转运功能的抑制来自对酶蛋白和膜结构影响两个方面,而非仅抑制酶蛋白本身的功能。     

cAMP在急性胃粘膜损害中的作用的研究

本研究应用ＲＩＡ法,观察了大鼠胃组织的ｃＡＭＰ含量与急性胃粘膜损害之间的关系。结果表明：（１）由消炎痛或失血性休克引起的急性胃粘膜损害时,胃组织ｃＡＭＰ含量明显降低;事先使用异搏定（１０ｍｇ／ｋｇ）可使胃组织ｃＡＭＰ含量明显增加,并使由消炎痛引起的急性胃粘膜损害相应减轻。（２）在正常情况下,胃窦、、胃体组织间ｃＡＭＰ含量并非相等,胃窦部组织ｃＡＭＰ含量高于胃体部组织,与此相应的是急性胃粘膜损害主要     

cAMP在急性胃粘膜损害中作用的研究

本研究应用ＲＩＡ法,观察了大鼠胃组织ｃＡＭＰ含量与急性胃粘膜损害之间的关系。结果表明：（１）由消炎痛或失血性休克引起的急性胃粘膜损害时,胃组织ｃＡＭＰ含量明显降低;事先使用异搏定（１０ｍｇ／ｋｇ）可使胃组织ｃＡＭＰ含量明显增加,并使由消炎痛引起的急性胃粘膜损害相应减轻。（２）在正常情况下,胃窦、胃体组织间ｃＡＭＰ含量并非相等,胃窦部组织ｃＡＭＰ含量高于胃体部组织,与此相应的是急性胃粘膜损害主要局限在胃体部。上述引起急性胃粘膜损害的诸因素均可使这种差别加大。５和１０ｍｇ／ｋｇ剂量的异搏定可使消炎痛所致急性胃粘膜损害的大鼠胃体部组织的ｃＡＭＰ含量明显增加,与胃窦部组织ｃＡＭＰ含量的差值变小,结果是胃体部粘膜的损害明显减轻。提示胃组织ｃＡＭＰ含量变化在急性胃粘膜损害中有一定的作用。     

钒酸钠、叠氮钠对小麦根细胞K~+吸收和质膜K~+-Mg~(2+)-ATPase的作用

Neurospora细胞膜质子泵(H~+-ATPase)专一性抑制剂钒酸钠,抑制小麦离体根K~+的吸收与H~+分泌,并抑制小麦根细胞膜-K~+-Mg~(2+)-ATPase活力。它对K~+吸收的抑制效应,可能是抑制质膜K~+-Mg~(2+)-ATPase活力的结果。而且在起抑制作用的时间上有明显地不同,表明钒酸钠对K~+、H~+在细胞膜中的通道影响不同。叠氮钠解链小麦根的呼吸,降低根细胞的ATP水平,但从实验开始就完全抑制小麦根K~+的吸收,对质膜K~+-Mg~(2+)-ATP-ase的活力没有影响。可能叠氮钠只阻止“载体”对K~+接受的过程。应用~86R_b+示踪的K~+吸收试验表明,钒酸钠对小麦根K~+吸收的抑制%,不为增加外部溶液K~+浓度而减低。增加底物ATP浓度,也不能减低钒酸钠对质膜-ATPase的抑制%。钒酸钠的抑制作用是非竞争性抑制。~3H-亮氨酸渗入试验表明钒酸钠对“载体”的合成没有干扰作用。VO_4~(3-)离子明显促进小麦根的呼吸,并提高根细胞的ATP水平,这种ATP水平的提高,可能是质膜-ATPase受到抑制,主动运输过程减弱的结果。     

胃粘膜Na~+-K~+-Mg~(2+)-ATPase在适应性细胞保护机制中的作用

本文观察了胃粘膜(Na~+-K~+-Mg~(2+))-ATPase在适应性细胞保护机制中的作用,并分析了其与内源性PG的关系。结果表明,哇巴因(一种(Na~+-K~+-Mg~(2+))-ATPase的抑制剂)可部分抑制胃蛋白酶150U(溶于0.1mol/L盐酸中)和20％乙醇的适应性细胞保护作用,并呈现明显的量效关系。用上述两种弱刺激灌胃后15min,胃粘膜(Na~+-K~+-Mg~(2+))-ATPase活力明显升高,也呈现明显的量效关系。预先给予消炎痛以抑制内源性PG的合成,则可阻断弱刺激所诱发的胃粘膜(Na~+-K~+-Mg~(2+))-ATPase活力的升高;若在此基础上再给予外源性PGE_2,又可解除消炎痛的阻断作用。这些结果说明,弱刺激通过内源性PG,进而促进胃粘膜(Na~+-K~+-Mg~(2+))-ATPase活力升高,使粘膜抵抗损伤的能力增强,可能是其保护作用的重要机制之一。     

胃(H++K+)-ATPase结构与功能的研究

胃(H⁺+K⁺)-ATPase属于生物膜的第二类质子泵(E₁E₂型),从生理角度它是胃酸分泌的质子泵。本文结合我们初步的研究结果:猪、大白鼠胃粘膜(H⁺+K⁺)-ATPase的纯化以及由消炎痛引起的急性胃粘膜病变与胃粘膜(H⁺+K⁺)-ATPase的关系等,对此酶在近十几年来它的纯化、结构、性质、催化机理,向胃腔分泌盐酸的功能及其调节和胃病变的分子机理等方面进行了简要的综述。     

吲哚乙酸对向日葵黄化幼苗下胚轴切段钾离子吸收的调节

IAA处理经适应性老化后的向日葵6～7日黄化幼苗的下胚轴切段,能显著促进它们的K~ 吸收和H~ 分泌。钒酸钠处理强烈地抑制K~ 吸收和H~ 分泌。IAA处理不改变K~ 吸收的V_(max)而使K_m明显变小。IAA处理显著促进在去离子水中的向日葵下胚轴切段的呼吸。KCl单独处理以较小的程度促进呼吸。IAA与KCl共同处理对呼吸的促进作用分别大于单独用IAA或KCI处理的促进作用,但小于这两个单独处理的促进作用之和。IAA促进的K~ 吸收并没有引起呼吸的进一步增加。IAA预处理向日葵下胚轴使从中提取的富含质膜制剂的K~ 刺激的ATP酶活力提高44.0％。在未经IAA预处理的富含质膜制剂中加IAA,K~ 刺激的ATP酶活力提高48.7％。IAA可以直接在质膜水平上促进向日葵下胚轴的质膜K~ 、Mg~(2 )—ATP酶活力。     

花生油对酒精所致胃粘膜损伤的保护作用

着重研究了花生油对无水酒精所引起的大白鼠胃粘膜损伤的保护作用,并对花生油作用的有效部位和机制作了初步探讨。     

Gastric (H+ + K+)-ATPase: modulation of the inhibitory properties of the novel potent antisecretagogue Ro 18-5364 by sulfhydryl reagents and nucleotides

The sulfoxide Ro 18-5364, a potential metabolite of the IND Ro 18-5362, is a powerful inhibitor of gastric mucosal (H+ + K+)-ATPase, decreasing enzymatic activity with an apparent Ki of 0.1 microM. Exposure of Ro 18-5364-treated gastric membranes to dithiothreitol fully restored (H+ + K+)-ATPase activity. ATP protected the enzyme against Ro 18-5364-induced inactivation of enzymatic activity. In addition, Ro 18-5364 inhibited vesicular proton uptake. In proton translocation experiments reduced lipoic acid methyl ester partially restored transport properties. Dithiothreitol and mercaptoethanol were without effect. The results are discussed with respect to the possible location of essential sulfhydryl groups for enzyme activity and proton transport.     

Sulphatide content in a membrane fraction isolated from rabbit gastric mucosal: its possible role in the enzyme involved in H+ pumping

The sulphatide content of vesicular membrane fraction from rabbit mucosal gastric microsomes was analyzed. This vesicular membrane fraction, in addition to a high sulphatide content, was enriched in an ouabain-insensitive (H+ + K+)-ATPase, a (Mg+2 + K+)-activated phosphatase, and a H+ pumping activity. The enzyme system involved in the process of acid secretion and the translocation of K+ was studied in these membrane preparations treated with arylsulphatase A, an enzyme that specifically hydrolyzes sulphatide. The results indicate that the breakdown of sulphatides of the vesicular membrane fraction inactivated both the (H+ + K+)-ATPase activity and the H+ pumping. Both activities were partially restored by the sole addition of sulphatide. The K+-stimulated ouabain-insensitive phosphatase activity, suggested as a partial reaction of the (H+ + K+)-ATPase sequence, was unaffected by arylsulphatase. These results suggest that sulphatides may play a function in the high activity binding site for K+ of the enzyme involved in H+ pumping.     

Inhibition of gastric (H+ + K+)-ATPase by the substituted benzimidazole, picoprazole

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.     

Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells.

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.     

The basal Mg2(+)-dependent ATPase activity is not part of the (H(+)+K+)-transporting ATPase reaction cycle.

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the 'stretched-out' version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.     

Gastric (H,K)-ATPase

Gastric acid secretion results from the activity of a specific ATPase, the (H+,K+)-ATPase. This enzyme, discovered in 1973, exchanges H+ for K+. It has two ATP binding sites, both involved in enzyme activity, whose affinities vary as a function of the H+ and K+ concentrations. Hydrolysis of ATP at the highest affinity site leads to the synthesis of a covalent aspartyl phosphate which accumulates in the absence of K+. The presence of this cation accelerates dephosphorylation resulting in the stimulation of ATPase (and PNPPase) activity. The structure of membranous (H+,K+)-ATPase is poorly defined. n-Octylglucoside solubilizes an active enzyme of 390-420 kDa which can be partly depolymerized using cholate. The monomer, characterized in SDS has a 95 kDa molecular mass and is inactive. In the presence of magnesium, (H+,K+)-ATPase catalyzes the active and neutral exchange of H+ for K+ at the expense of ATP. In the absence of ATP, (H+,K+)-ATPase acts as a passive transporter exchanging K+ for K+ at maximal rate and H+ for K+ at a 20 times slower rate.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Gastric acid secretion in the lizard. Ionic requirements and effects of inhibitors.

1. The effects of ion substitution and various inhibitors on the transmucosal potential, short circuit current, mucosal resistance and acid secretion of the lizard gastric mucosa, incubated in an Ussing chamber, have been determined. 2. Ion substitution experiments indicate that the serosal potential step consists of a combined C1- and K+ diffusion potential, and that the mucosal potential step is Na+ dependent and behaves primarily as a Na+ diffusion potential. 3. Experiments with ouabain indicate that the major (Na+, K+)-ATPase activity responsible for maintenance of cation gradients is located on the serosal side of the mucosal cells, and that this pump activity is non-electrogenic. 4. Experiments with amiloride indicate that a passive sodium influx on the mucosal side is essential for the maintenance of the transmucosal potential and short circuit current. 5. Acid secretion requires the presence of sodium and chloride on the serosal side and the maintenance of a high intracellular potassium level through the (Na+, K+)-ATPase system. 6. The effects of acetazolamide and thiocyanate are compatible with an involvement of carbonic anhydrase and anion-dependent ATPase in acid secretion. 7. Upon initiation of acid secretion the serosal membrane permeability for chloride increases and that for potassium decreases.