Aminopyrine Accumulation Research Articles

Characterization of the effects of adenosine, adenosine 5′-triphosphate and related purines on acid secretion in isolated rabbit gastric glands

The influence of adenosine, AMP, ADP, ATP, the adenosine analogue l-PIA and the ATP analogue β,γ-methylene ATP, on gastric acid secretion, as measured by the aminopyrine accumulation method, in resting and histamine-stimulated glands isolated from rabbit gastric mucosa was studied. In resting glands, adenosine and its analogue l-PIA (10 μ m-1 m m) caused significant concentration-related increases of the basal H + secretion, whereas no changes were obtained in response to the other purines tested. In histamine-stimulated glands, adenosine, l-PIA and AMP (10 μ m-1 m m) induced concentration-related increases of the H + secretory rate, whereas ATP, β,γ-methylene ATP and ADP (10 μ m-1 m m) produced concentration-related decreases of the H + raised rate. The rank order of potency of the purine compounds in increasing the stimulated H + secretion was: adenosine > l-PIA ≫ AMP, and in decreasing it was: ATP> ADP> β,γ-methylene ATP. The stimulatory responses to adenosine were inhibited by theophylline (10 μ m–100 μ m) and caffeine (10 μ m-1 m m); whereas, the inhibitory responses to ATP were significantly reduced by the well known prostaglandin synthesis inhibitor indomethacin (1 μ m–100 μ m). From the results it is concluded that in isolated rabbit gastric glands, purine compounds are effective modulators of the gastric H + secretory process. The pattern of purine activity obtained suggests that the stimulatory responses, inhibited by methylxanthines, may be mediated via P 1-purinoceptors, while the inhibitory responses, reduced by indomethacin, may be mediated via P 2-purinoceptors.

Dependence of cellular energy metabolism on the intracellular pH of isolated rabbit parietal cells.

The cellular energy metabolism under various extracellular pH (pHe) was investigated in parietal cells isolated from rabbit gastric mucosae. Activity of parietal cells was checked by measuring effects of histamine and db-cAMP on the accumulation of aminopyrine in the intracellular canaliculi. The relationship between the intracellular pH (pHi) and pHe was determined by using a fluorescence probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in suspensions of isolated parietal cell. The pHi was a linear function of pHe and it changed by 0.63 pH unit per 1.0 unit change in pHe. The internal [H+] was equal to the external [H+] at approximately pH 7.0. The respiratory rate of the cell depended on pHi. Values of [ATP] + [ADP] and intracellular inorganic phosphoric acid [Pi] were almost independent of pHi, whereas the [ATP]/[ADP] ratio decreased with increase in pHi. In addition, the standard free energy of ATP(delta Go'ATP) decreases as pHi increases. The calculated available free energy of ATP, which is the sum of delta Go'ATP and -RTlog[ATP]/([ADP] [Pi]), was independent of pHi in the range from pHi 6.2 to 7.6 and its value was about -11.7 kcal/mol. Furthermore, [ATP], [ADP], and [Pi] in the intracellular space were not affected by the presence of histamine or dibutyryl cAMP in media. It was indicated that the available free energy of ATP did not change in the transformation from resting to acid-secreting states, and the phosphorylation potential might have an important role in control of homeostasis in energy metabolism.

Direct Hyposmotic Stimulation of Gastric Acid Secretion

Gastric glands isolated from rabbit stomach were incubated in isosmotic medium or media made hyposmotic by 50-100 mOsm/kg. As indicated by radiolabeled aminopyrine accumulation, acid secretion was nearly 3 times greater in 200 mOsm/kg hyposmotic than in isosmotic medium after a 30-min incubation. The hyposmotic stimulation appeared within 2 min, peaked at 10-15 min and declined almost to the isosmotic control by 45 min. As estimated by the wet weight corrected for inulin extracellular space, the intracellular water of the glands also peaked at 15 min and returned to the isosmotic norm by 45 min. Hyposmotic stimulation of acid secretion directly involved the parietal cell, since parietal cells obtained from gastric glands were also stimulated. That the hyposmotic response was direct was indicated by omeprazole inhibition of aminopyrine accumulation in hyposmotic medium.

P-262 - Effects of IT-066, a new H2-receptor antagonist, on [14C]aminopyrine accumulation in isolated rabbit parietal cells

P-262 - Effects of IT-066, a new H2-receptor antagonist, on [14C]aminopyrine accumulation in isolated rabbit parietal cells

Calcium involvement in the muscarinic response of the gastric parietal cell

The influence of extracellular Ca 2+ on the mediation of carbachol stimulation in isolated rabbit gastric parietal cells was studied. Removing Ca 2+ from extracellular medium caused a 42% decrease of the aminopyrine accumulation due to carbachol with the same EC 50 value (∼ 5 μM). A short time depletion in extracellular calcium suppressed the carbachol-dependent Ca 2+ influx without affecting Ca 2+ release from internal stores (fura-2 measurements). Similarly, the production of inositol phosphates under cholinergic stimulation was reduced by 29%. A rapid increase in Ins(1, 4, 5)P 3 was obtained 5 s after carbachol stimulation, and this increase was not changed in Ca 2+-dependent medium. In contrast, a 20 min incubation with carbachol caused a 50% reduction in both basal and carbachol-stimulated inositol phosphate accumulations. In conclusion, phospholipase C activation, intracellular Ca 2+ release and aminopyrine accumulation were sequentially observed following carbachol stimulation of the isolated gastric parietal cell and extracellular calcium contributed to sustain this acid secretory response.

Enhancing effect of pyrrolidone derivatives on the transdermal penetration of sulfaguanidine, aminopyrine and sudan III.

The enhancing effect of pyrrolidone derivatives on the percutaneous penetration of sulfaguanidine, aminopyrine and sudan III was investigated using in vitro technique and excised rat skin. 1-Methyl (MP), 1-hexyl (HP) and 1-lauryl-2-pyrrolidone (LP) were used as penetration enhancers. Aminopyrine showed high penetration through skin although sulfaguanidine and sudan III showed little penetration. Pyrrolidone derivatives enhanced their penetrations. Especially HP and LP enhanced the penetration of sulfaguanidine to a high extent. Sudan III was not detected in the receptor phase regardless of the presence of enhancer. Pyrrolidone derivatives significantly increased the skin accumulation of sulfaguanidine, aminopyrine and sudan III. Penetration of pyrrolidone derivatives was also determined. MP and HP showed high penetrations. LP was not detectable in the receptor phase. MP, HP and LP showed high skin accumulations. These results suggested the usefulness of pyrrolidone derivatives as percutaneous penetration enhancers.

Muscarinic receptors mediating acid secretion in isolated rat gastric parietal cells are of M3 type

Five subtypes of muscarinic receptors have been identified by pharmacological and molecular biological methods. The muscarinic receptor subtype mediating acid secretion at the level of the parietal cell was unknown. Therefore, this study was performed to characterize muscarinic receptors on rat gastric parietal cells using the 3 subtype-selective antagonists hexahydrosiladifenidol and silahexocyclium, which have high affinity for glandular M3 subtypes, and AF-DX 116, which has high affinity to cardiac M2 receptors. The affinity of these antagonists was determined by radioligand binding experiments. In addition, their inhibitory potency on carbachol-stimulated inositol phosphate production was investigated. Inhibition of carbachol-stimulated aminopyrine uptake was used as an indirect measure of proton production. Both M3 antagonists, hexahydrosiladifenidol and silahexocyclium, had nanomolar affinities for parietal cell muscarinic receptors and potently antagonized inositol phosphate production with nanomolar Ki values. Silahexocyclium similarly antagonized aminopyrine accumulation while hexahydrosiladifenidol behaved as a noncompetitive antagonist. AF-DX 116 was a low-affinity ligand and a weak competitive antagonist at parietal-cell muscarinic receptors. It was concluded that muscarinic M3 receptors mediate acid secretion probably by activation of the phosphoinositide second messenger system in rat gastric parietal cells.

Effect of adenosine and adenosine analogs on [14C]aminopyrine accumulation by rabbit parietal cells

Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on [14C]aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. [14C]Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated [14C]aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on [14C]aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased [14C]aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on [14C]aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.

Inhibition of acid formation and stimulation of somatostatin release by cholecystokinin-related peptides in rabbit gastric glands.

1. The purpose of the present study was to investigate the role of somatostatin in the inhibition of acid production induced by caerulein and cholecystokinin (CCK) in isolated rabbit gastric glands. Acid production was estimated by the aminopyrine technique. 2. Exogenous somatostatin 14 and somatostatin 28 (10(-7) M) reduced to a similar extent the aminopyrine uptake produced by 5 x 10(-5) M-histamine during the course of 40 min incubation. 3. Significant inhibition of histamine-stimulated aminopyrine accumulation occurred at a somatostatin 14 concentration of 10(-9) M. 4. Caerulein and CCK octapeptide (10(-13)-10(-7) M) were found to release somatostatin from isolated gastric glands in a dose-dependent manner. The dose-response relationships for somatostatin release and inhibition of aminopyrine uptake were similar. Thus, the half-maximal dose approximations for somatostatin release and inhibition of aminopyrine uptake were 0.5 and 1.4 x 10(-9) M respectively for CCK octapeptide and 0.9 and 2.5 x 10(-11) M for caerulein. Heptadecapeptide gastrin proved to be a very poor releaser of somatostatin in the system used. The CCK octapeptide-induced somatostatin release was time dependent and the concentrations of somatostatin that accumulated in the incubation medium were similar to those of exogenous somatostatin that were needed to evoke inhibition. 5. The present results support the concept that cholecystokinin inhibits gastric acid secretion by releasing somatostatin from endocrine-like cells in the gastric mucosa. It is suggested that cholecystokinin-related peptides may play a physiological role in inhibiting gastric acid secretion. A similar role for gastrin is not supported by the present study.

A micromethod for the assay of cellular secretory physiology: Application to rabbit parietal cells

A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [ 14C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [ 14C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [ 14C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.

Pancreastatin: A novel peptide inhibitor of parietal cell signal transduction

The direct inhibition of secretion by pancreastatin was investigated in rabbit isolated parietal cells. Pancreastatin exerted no influence on basal aminopyrine uptake. Pancreastatin inhibited histamine stimulated aminopyrine uptake through a decrease in intracellular cAMP. Pancreastatin inhibition of histamine stimulated uptake was blocked in the presence of pertussis toxin. Pancreastatin also inhibited the carbachol stimulated increase in aminopyrine accumulation. However, the effects of pancreastatin on carbachol stimulation were not reversed by pertussis toxin. Pancreastatin did not alter the carbachol induced increase in cytosolic free calcium. Thus, pancreastatin appears to inhibit parietal cell signal transduction at multiple points along the second messenger pathways.

Carbachol-induced protein phosphorylation in parietal cells: regulation by [Ca2+

1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), an intracellular calcium [( Ca2+]i) chelator, was used to investigate the role of [Ca2+]i in acid secretory activity and protein phosphorylation in parietal cells from rabbit. Chelation of extracellular calcium [( Ca2+]o) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not prevent the initial carbachol-induced elevation of [Ca2+]i as measured with the fluorescent Ca2+ probe, fura-2, and only partially inhibited [14C]-aminopyrine (AP) accumulation, an indirect indicator of acid secretory activity. [Ca2+]i chelation with BAPTA/AM eliminated carbachol-stimulated increases in [Ca2+]i and AP accumulation but only transiently reduced histamine stimulation of AP accumulation. Carbachol increased phosphorylation of a 36-kDa, pI approximately 7 protein (pp36) and transient phosphorylation of a 28-kDa, pI approximately 5 protein (pp28), whereas histamine increased phosphorylation of 40-kDa, pI approximately 6.5 (pp40) and 27-kDa, pI approximately 6.2 (pp27) proteins. Phosphorylation of pp36 and pp28 were mimicked by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, ionomycin, respectively. Two other phosphoproteins with molecular weights of 66,000 and pIs of 5.7 and 5.9 were also phosphorylated in response to TPA and carbachol. Chelation of [Ca2+]i and [Ca2+]o blocked carbachol-induced phosphorylation of pp28 and pp36 and ionomycin phosphorylation of pp28 but not TPA-stimulated phosphorylation of pp36 or the two pp66s or histamine-stimulated phosphorylation of pp27 or pp40. Chelation of [Ca2+]i alone did not block increases in [Ca2+]i or phosphorylation of pp28 in response to ionomycin. Both pp28 and pp36 were localized in both microsomal and cytosolic fractions of cells, which suggests involvement in cytoskeleton-membrane interactions. These phosphoproteins could be common elements of Ca2+-dependent stimulus-secretion coupling as similar proteins were phosphorylated by carbachol and cholecystokinin (CCK) in chief cells. Based on data with TPA and ionomycin, both protein kinase C and an as yet unidentified Ca2+-dependent protein kinase(s) appear to be activated upon stimulation with cholinergic agonists and CCK.

Role of Ca 2+ in H + transport by rabbit gastric glands studied with A23187 and BAPTA, an incorporated Ca 2+ chelator

The role of Ca 2+ in stimulation of H + gastric secretion by cAMP-dependent and -independent secretagogues was studied in isolated rabbit glands using Ca 2+ ionophore, A23187, and an intracellular Ca 2+ chelator (BAPTA, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid) incorporated as its acetoxymethyl ester (BAPTA-AM). Acetylcholine (ACh), tetragastrin (TG), histamine and forskolin induced a transitory increase of intracellular Ca 2+ concentration, [Ca 2+] i, measured in gastric glands loaded with Ca 2+-sensitive dye fura-2, and provoked an acid secretory response evaluated with aminopyrine accumulation ratio (AP ratio). The Ca 2+-ionophore A23187 also induced an increase in [Ca 2+] i and in AP ratio. cAMP-dependent secretagogues were more potent stimulants of acid secretion than cAMP-independent secretagogues. cAMP analogue, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-BR-cAMP) induced an increase in AP ratio without modifying [Ca 2+] i. BAPTA-AM (5–25 μM) induced a transient decrease of resting [Ca 2+] i which returned to basal level due to extracellular Ca 2+ entry. Increases in [Ca 2+] i produced by ACh and TG were abolished by BAPTA and those produced by Ca 2+ ionophore A23187 were partially buffered. BAPTA inhibited in a dose-dependent manner H + secretion induced by cholinergic and gastrinergic stimulants in the presence of cimetidine. A23187 increased the AP ratio to values similar to those obtained with ACh or TG and was not inhibited by BAPTA. BAPTA partially inhibited (40%) the increase in AP ratio induced by forskolin and histamine inspite of the complete inhibition of the Ca 2+ response. BAPTA did not inhibit the response to 8-BR-cAMP. BAPTA inhibition of forskolin stimulation was reversed by A23187 and the response was potentiated. These results indicate that ACh and TG response are completely dependent on an increase of [Ca 2+] i. The response to cAMP-dependent agonists histamine and forskolin depend both on Ca 2+ and cAMP. For forskolin stimulation the response may be the result of a potentiation between Ca 2+ and cAMP.

Interaction of signal transduction pathways in mediating acid secretion by rat parietal cells

To examine the role of protein kinase C (PKC) on the acid secretory activity of isolated rat parietal cells, histamine-and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated [14C]aminopyrine accumulation was determined in the presence of agents that redistribute PKC activity to plasma membranes. Phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetylglycerol (OAG), and phospholipase C inhibited, in a dose-dependent fashion, histamine- and DBcAMP-stimulated [14C]aminopyrine accumulation. Because PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) reversed the effect, the results suggest that inhibition of histamine- or DBcAMP-stimulated [14C]aminopyrine accumulation induced by PMA, OAG, or phospholipase C was caused by increased activity of PKC in plasma membrane. To determine where in the cascade of events PKC inhibits acid secretion, histamine-, cholera toxin-, and forskolin-stimulated [14C]aminopyrine accumulation was measured with or without PMA. Because the percent of inhibition by PMA of [14C]aminopyrine accumulation was similar with the three secretagogues, the results suggest that PKC inhibits acid secretion at a point beyond adenosine 3',5'-cyclic monophosphate (cAMP) production. This was supported by the fact that PMA had no effect on histamine-stimulated production of cAMP and by the finding that activation of PKC had the same effect on histamine- or DBcAMP-stimulated [14C]aminopyrine accumulation. Histamine and DBcAMP inhibited PKC activity, suggesting a reciprocal interaction between PKC and histamine-triggered signal transduction pathway.

Pharmacological properties of the hydroxylated histamine, (±)-4(5)-(2-amino-1-hydroxyethyl)-imidazole

The pharmacological effects of the beta-hydroxylated histamine, 4(5)-(2-amino-1-hydroxyethyl)-imidazole, on smooth muscle contraction of the ileum (H1-receptor activity) and gastric acid secretion (H2-receptor activity) of the guinea-pig were investigated and compared with those of histamine. Although beta-hydroxy histamine contracted the ileum with the same maximal response as histamine, the concentration response curve was shifted to the right by approximately three orders of magnitude. At submaximal concentrations, co-administration of beta-hydroxy histamine with histamine revealed only additive effects. This H1-activity was competitively inhibited by diphenhydramine. Similarly, the hydroxylated analogue also increased intracellular cyclic AMP level and [14C] aminopyrine accumulation as a marker of acid secretion in the parietal cells. However, the EC50 was approximately ten fold that of histamine. This H2-receptor activity was inhibited completely by cimetidine. These results suggest that beta-hydroxy histamine possesses nearly full intrinsic activities at both H1 and H2-receptors and that the introduction of a hydroxyl group at the beta-carbon reduces and dissociates these activities.

Intracellular Ca requirements for stimulus-secretion coupling in parietal cell

The role of cytosolic free Ca (Cai) in stimulating acid production by the parietal cell in response to the secretagogues carbachol (carb), histamine (hist), and dibutyryl adenosine 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine (DBcAMP + IBMX) was evaluated. Microfluorimetry with fura-2 was used to measure Cai in single parietal cells within intact rabbit gastric glands. Acid production was determined in parallel experiments by monitoring the accumulation of [14C]aminopyrine (AP) in suspensions of glands. Carb increased peak Cai levels to 1 microM in a dose-dependent manner [concentration for half-maximal response (K0.5) = 8 microM] that correlated well with the dose dependence of carb-stimulated AP accumulation (K0.5 = 18 microM). The Ca chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was used to attenuate secretagogue-induced Cai increases. Incubating glands for 10 min with 1 and 10 microM BAPTA/AM caused resting Cai to decrease from 119 to 93 and 80 nM, respectively. BAPTA/AM, 1 and 10 microM, blocked carb-stimulated increases in Cai by 60 and 90% and AP accumulation by 50 and 90%. Hist, which increases cytosolic cAMP, caused small and relatively infrequent increases in Cai. Even so, hist-stimulated AP accumulation was inhibited 8 and 40% by 1 and 10 microM BAPTA. DBcAMP had no effect on Cai, and AP accumulation caused by DBcAMP was unaffected by the concentrations of BAPTA tested. These data suggest that carb requires an increase in Cai as a secretory signal. Hist also exhibited some Cai dependence, which may be attributable to either a small increase in Cai or the necessity of having a specific basal level of Cai. A Cai requirement for DBcAMP-stimulated acid secretion was not detected. Thus the parietal cell possesses both Ca-dependent and Ca-independent stimulatory pathways, and at least one secretagogue (hist) may utilize both pathways.

Inhibition of Acid Formation in Rabbit Parietal Cells by Prostaglandins Is Mediated by the Prostaglandin E2 Receptor

The affinities of seven natural and synthetic prostaglandins [PGE2; 16,16-dimethyl PGE2; iloprost (stable prostacyclin analogue); PGF2 alpha; PGD2; BW245c (stable PGD2 analogue); and U46619 (stable thromboxane analogue)] to the PGE2 binding site of rabbit gastric mucosa were determined by measuring [3H]PGE2 displacement from its high-affinity plasma membrane binding sites. In parallel, the potency of each prostaglandin in inhibiting acid generation in vitro was determined by measuring the inhibition of histamine-stimulated [14C]aminopyrine accumulation in rabbit parietal cells prepared by enzymatic dispersion and enriched by counterflow elutriation. All seven prostaglandins displaced [3H]PGE2 and inhibited histamine-stimulated [14C]aminopyrine accumulation in a concentration-dependent manner. For all tested prostaglandins, the IC50 values were in excellent agreement for both variables measured. It is concluded that (a) a PGE2 receptor is localized on the parietal cell and mediates inhibition of acid formation by all prostaglandins and (b) the different in vitro antisecretory potencies of prostaglandins can be attributed to their different affinities to this PGE2 receptor.

Inhibition of H+, K+-ATPase by Methyl(E)-2-(3, 4-Dimethoxystyryl)-Benzimidazole-4-Carboxylate (ALE-36)

ALE-36, as well as omeprazole and SCH 28080, markedly inhibited the [14C]aminopyrine (AP) accumulation induced by dibutyryl cyclic AMP (dbcAMP) and H+,K+-ATPase activity in a concentration-dependent manner. The inhibitory effect of omeprazole on the dbcAMP-induced [14C]AP accumulation was reversed by treatment with beta-mercaptoethanol, but those of ALE-36 and SCH 28080 were not. ALE-36 and SCH 28080 did not inhibit dog renal Na+,K+-ATPase activity, while omeprazole and ouabain did inhibit this enzyme activity. These results suggest that the inhibitory action of ALE-36 on acid secretion is due to the specific inhibition of gastric H+,K+-ATPase, the manner being different from in the case of omeprazole.

Local Anesthetics (Benzyl Alcohol, Lidocaine, Procainamide) Inhibit Aminopyrine Accumulation in Isolated Rat Parietal Cells

The present studies were designed to examine the effect of local anesthetics (benzyl alcohol, lidocaine, and procainamide) on the secretory response of parietal cells to histamine, dbcAMP, and carbachol. Studies were performed in vitro using isolated cells from rat stomachs, and acid production was determined by 14C-aminopyrine accumulation. In addition, the (H(+)-K+)-ATPase activity of microsomal vesicles isolated from parietal cells was determined. Lower concentrations of the drugs studied increased the basal aminopyrine accumulation and potentiated the secretory response of parietal cells to histamine and dbcAMP. At higher concentrations local anesthetics progressively inhibited both the basal 14C-aminopyrine accumulation and that stimulated by histamine, dbcAMP or carbachol. While a low concentration of local anesthetics increased gastric microsomal (H(+)-K+)-ATPase activity, higher concentrations inhibited enzyme activity to about 80% of those activities found in resting parietal cells. We conclude that increased aminopyrine accumulation may reflect the activation of membrane-bound enzyme(s) involved in the cAMP-dependent signal transduction pathway mediating acid secretion by parietal cells. In turn, it is possible that the inhibition of aminopyrine accumulation by local anesthetics at higher concentrations can relate to two different mechanisms: (1) the nonspecific effect of local anesthetics that causes simple proton neutralization (as weak bases), and (2) to a minor extent their inhibitory effect on proton pump activity.

Sites of action of protein kinase C on secretory activity in rat parietal cells

Sites at which the calcium-sensitive phospholipid-dependent protein kinase, protein kinase C, may influence acid secretion have been investigated in rat isolated parietal cells. In both crude and enriched preparations of parietal cells incubated in a medium containing 100 mM K+, the activators of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-glycerol (OAG), produced a dose-dependent stimulation [half-maximally effective concentration (EC50) values of 1 nM and 70 microM, respectively] of aminopyrine accumulation, an index of the sequestration of acid in the cell. In a medium containing 4.5 mM K+, and with no added secretagogues, TPA and OAG did not affect aminopyrine accumulation. Histamine-stimulated aminopyrine accumulation was inhibited by TPA [half-maximally effective inhibitory concentration (IC50) of 2.9 nM]. TPA reduced the histamine-stimulation of the adenosine 3',5'-cyclic monophosphate (cAMP) content of parietal cells (47% inhibition at 100 nM TPA) but also inhibited aminopyrine accumulation at or distal to cAMP-dependent protein kinase. Activators of protein kinase C can produce multiple effects on secretory activity in the rat parietal cell.