AP Uptake Research Articles

The use of anticoagulants in the management of atrial fibrillation among general practices in England

ObjectivesTo investigate the use of oral anticoagulants (AC) and antiplatelet agents (AP) in the management of atrial fibrillation (AF) among patients in primary care in England.DesignEpidemiological study.Setting1857 general practices in...

Antisecretory actions of Baccharis trimera (Less.) DC aqueous extract and isolated compounds: Analysis of underlying mechanisms

Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models. To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera. AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H(+), K(+)-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions. Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4°C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 μM) and F9 (10-300 μg/ml) decreased the basal [(14)C]-AP uptake by 50 and 60% of control (Ratio=6.2±1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 μg/ml) decreased the [(14)C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [(14)C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [(14)C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [(14)C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H(+), K(+)-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro. The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract.

Mode de révélation inhabituel d’un syndrome de Sjögren primitif

Aprotinin (Ap), a basic polypeptide with a molecular weight of 6500, is filtered at the glomerular membrane without steric restriction and is completely absorbed by the proximal tubule cells. Here Ap is broken down to amino acids, but no breakdown products enter the peritubular circulation during the first 20 min following an intravenous injection. These properties have recently been exploited for measurement of local glomerular filtration rate, based on the assumption that the proximal tubular uptake site is located at the level of the filtering glomerulus. To evaluate that assumption we have now made serial autoradiographs of the rat kidney 20 min after intravenous injection of 2–750 μg of 125I-Aprotinin. With all doses the percent 125I-containing proximal tubular transections were about 50 in the outer and middle cortex and 35 in the inner third. We interpret these numbers to mean that all filtered Ap is taken up in the first two thirds of the proximal convoluted tubular length and does not reach the pars recta. Since the proximal tubule on average is located more superficial than its glomerulus, measurement of local Ap uptake will tend to overestimate glomerular filtration rate in outer layers of the cortex. Quantitative estimate of this “displacement” will be presented in a companion article.

Aprotinin uptake in the proximal tubules in the rat kidney I.: Length of proximal tubular uptake segment

Aprotinin (Ap), a basic polypeptide with a molecular weight of 6500, is filtered at the glomerular membrane without steric restriction and is completely absorbed by the proximal tubule cells. Here Ap is broken down to amino acids, but no breakdown products enter the peritubular circulation during the first 20 min following an intravenous injection. These properties have recently been exploited for measurement of local glomerular filtration rate, based on the assumption that the proximal tubular uptake site is located at the level of the filtering glomerulus. To evaluate that assumption we have now made serial autoradiographs of the rat kidney 20 min after intravenous injection of 2–750 μg of 125 I -Aprotinin. With all doses the percent 125 I -containing proximal tubular transections were about 50 in the outer and middle cortex and 35 in the inner third. We interpret these numbers to mean that all filtered Ap is taken up in the first two thirds of the proximal convoluted tubular length and does not reach the pars recta. Since the proximal tubule on average is located more superficial than its glomerulus, measurement of local Ap uptake will tend to overestimate glomerular filtration rate in outer layers of the cortex. Quantitative estimate of this “displacement” will be presented in a companion article.

Platelet-activating factor stimulates gastric acid secretion in isolated rabbit gastric glands

We examined the effect of platelet-activating factor (PAF) on gastric acid secretion by isolated rabbit gastric glands as determined by [14C]aminopyrine ([14C]AP) uptake. PAF, histamine, and carbachol time- and concentration-dependently stimulated [14C]AP uptake, with estimated half-maximal effective concentrations of 60 pM, 0.25 microM, and 0.1 microM, respectively. PAF-induced [14C]AP uptake was inhibited by the specific PAF antagonists BN-50727 and SR-27417 and by the proton pump inhibitors omeprazole and lansoprazole. However, the H2-receptor antagonist famotidine had no effect. Buffering extracellular Ca2+ by ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid resulted in a shift to the right of the time-course effect of PAF without altering the maximal response, whereas buffering intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid 2-acetoxymethyl ester, as well as blocking Ca2+ channels by verapamil, inhibited PAF-induced [14C]AP uptake. Intracellular Ca2+ concentration in isolated rabbit gastric glands, as measured by fura 2-acetoxymethyl ester, concentration-dependently increased in response to PAF, to a maximum of 1.5-fold for 0.1 microM. These results suggest that PAF stimulates gastric acid secretion via specific receptors activating intracellular Ca2+ mobilization, which could be located on the parietal cells.

Secretagogue-induced [14C]aminopyrine uptake in isolated equine parietal cells

Summary Equine oxyntic mucosal cells were obtained by sequential exposure to pronase and collagenase. Acid production by parietal cells was assessed by uptake of [14C]aminopyrine (ap), a weak base that accumulates in intracellular acidic spaces. Incubation for various times revealed a maximal ap uptake at 10 minutes for histamine and carbachol. Similar secretagogue responses were observed for parietal cells from the mucosal cell preparation or after enrichment by elutriation. Histamine and isobutyl-methylxanthine (ibmx) stimulated AP uptake with a dose-dependent response and maximal effective concentration of 100 μM. Carbachol, 1 to 100 μM, and pentagastrin (pg), 1 to 1,000 μM, were ineffective stimulants of ap uptake. The ap uptake values for 100 μM ibmx, 1 μM carbachol, or 100 nM pg were 77 ± 6%, 50 ± 3-2%, and 40 ± 4.5%, respectively, of that observed with maximal stimulation by 100 μM histamine (mean ± sem, n = 4 to 14). Uptake of ap by nonstimulated control cells was 36 ± 3.6% of maximal histamine stimulation. The ap accumulations during control conditions and after stimulation with 100 μM histamine and IBMX, 1 μM carbachol, or 100 nM pg were 1.18 ± 0.39, 2.81 ± 0.85, 1.93 ± 0.48, 1.44 ± 0.36, and 1.23 ± 0.33 pmol of ap/105 parietal cells, respectively. Individual histamine dose-response curves were shifted to the right by increasing ranitidine and cimetidine concentrations (0.1 to 50 μM). These results indicate that isolated equine parietal cells are maximally stimulated by histamine and minimally stimulated by carbachol and pg.

Mechanism and kinetics of transcellular transport of a new beta-lactam antibiotic loracarbef across an intestinal epithelial membrane model system (Caco-2).

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H+ gradient-dependent pathway (Km = 0.789 mM, and Jmax = 163 pmol/min per cm2) is relatively "high affinity" and "low capacity" compared to H+ gradient-independent pathway (Km = 8.28 mM, and Jmax = 316 pmol/min per cm2). In addition, TT of LOR in the presence of a H+ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H+ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H+ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H+ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.

Ezrin-calpain I interactions in gastric parietal cells.

Gastric ezrin, a membrane-cytoskeletal linker with sequence homology to talin and erythrocyte band 4.1, has been associated with the remodeling of parietal cell apical membrane that occurs with adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase stimulation. Here we examine the interrelationship between parietal cell ezrin and Ca(2+)-dependent protease activity. Addition of Ca2+ to sonicated gastric gland preparations rendered a relatively selective proteolysis of the 80-kDa ezrin, accompanied by the appearance of a 55-kDa breakdown product. Ca(2+)-dependent proteolysis of ezrin was blocked by E64, a cysteine protease inhibitor, or calpastatin, indicating calpain as the responsible protease. Degradation of ezrin in intact gastric glands was achieved by varying extracellular [Ca2+] and [ionomycin]. Ezrin degradation in situ was rapid and relatively selective, although Ca(2+)-dependent degradation of some spectrin-like bands was also observed. The effect of activated calpain I on parietal cell function was assessed by probing the secretory response to histamine stimulation using [14C]aminopyrine uptake, along with parallel measurements of calpain activity, over a wide range of ionomycin. Activation of calpain, as evidenced by loss of parietal cell ezrin, was correlated with decreased AP uptake by stimulated gastric glands, supporting a role for ezrin in the oxyntic secretory process. The calpain-ezrin interaction established here, and the similarities of calpain with talin and erythrocyte band 4.1, suggest a common feature to this family of ezrin/band 4.1/talin proteins that have been implicated in membrane-cytoskeletal association.

Relationship between inositol 1,4,5-trisphosphate mass level and [ 14C]aminopyrine uptake in gastrin-stimulated parietal cells

The relationship between gastrin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) content and [ 14C]aminopyrine ([ 14C]AP) uptake (an index of in vitro acid secretion) was investigated in a population of highly enriched rabbit parietal cells (90 ± 5%). Gastrin induced a rapid rise in Ins(1,4,5)P 3 content which was maximal within 15 s of stimulation (2- to 2.5-fold basal level) followed by a rapid decrease within 30 s; a high Ins(1,4,5)P 3 level could also be observed after a longer time of hormone stimulation (180 s). Gastrin dose-dependently induced Ins(1,4,5)P 3 accumulation and [ 14C]AP uptake; both dose-response curves were similar (EC 50 ≈ 0.1 nM). Furthermore, L-365, 260 (3-(acylamino)benzodiazepine), a selective gastrin/ CCK-B receptor antagonist, dose-dependently inhibited Ins(1,4,5)P 3 production and [ 14C]AP accumulation induced by 10 nM gastrin with a similar potency (IC 50 ≈ 1–2 nM). These results led us to conclude that Ins(1,4,5)P 3 is involved in gastrin-stimulated acid secretory activity of gastric parietal cells.

Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells

The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[ 3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [ 3H]inositol trisphosphate ([ 3H]InsP 3) accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (< 10 μM), potentiated gastrin-induced [ 3H]InsP 3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [ 3H]InsP 3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15–50% reduction in gastrin-induced [ 14C]aminopyrine ([ 14C]AP) uptake (an index of in vitro acid secretion) and [ 3H]inositol phosphate ([ 3H]InsP) accumulation. A decrease in the accumulation of the different [ 3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [ 14C]AP uptake ( EC 50 values: 0.125 ± 0.045 nM without PTx and 1.05 ± 0.63 nM with PTx), for [ 3H]InsP accumulation ( EC 50 values: 0.16 ± 0.08 nM without PTx and 1.56 ± 0.58 nM with PTx) and [ 125I]gastrin binding ( IC 50 values: 0.247 ± 0.03 nM without PTx and 2.38 ± 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [ 14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive ‘G i-like’ protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of 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.

The effect of arachidonic acid and its metabolites on acid production in isolated human parietal cells.

The effect of arachidonic acid and its metabolites on the histamine-stimulated acid production in human isolated parietal cells provenient from endoscopic biopsies was examined. 14C-aminopyrine (14C-AP) accumulation in the parietal cells was used for evaluation of acid production. Histamine dose-dependently increased AP uptake. Histamine stimulation (taken as 100% at 10(-5) M) was significantly inhibited by prostaglandin (PG) E2 to 66 +/- 7% at 10(-8) M, 42 +/- 8% at 10(-6) M, and 13 +/- 10% at 10(-4) M (mean +/- SEM, n = 10). PGF2 alpha, PGD2, and PGI2 showed significant inhibitory effects only at very high concentrations (10(-5)-10(-4) M). Leukotriene (LT) B4 and LTC4 were without effect. The basal acid production (taken as 0%) was lowered significantly by 10(-6) M arachidonic acid to -20 +/- 7.4% (p less than 0.02, n = 10), and the histamine-stimulated (10(-6) M) acid production from 100% to 64 +/- 7.2% (p less than 0.001, n = 10). Aspirin (10(-3) M) increased basal (45 +/- 9.6%, p less than 0.001, n = 10) and histamine-stimulated (10(-6) M) acid production (164 +/- 16.3%, p less than 0.001). It is concluded that PGE2, the major product from arachidonic acid metabolism in the human gastric mucosa, is a significant inhibitor of the histamine-stimulated human parietal cell and may, in humans, play a role as a local physiologic inhibitor of acid secretion.

Inhibition of acid formation by epidermal growth factor in the isolated rabbit gastric glands.

The effects of epidermal growth factor (EGF) on basal and stimulated (with histamine, dibutyryl cyclic AMP, and high concentrations of K+) acid formation have been studied in isolated glands from the rabbit gastric mucosa. The changes in the accumulation of [14C]aminopyrine [14C]AP have been used as an indirect measurement of acid production in the glands. Unstimulated gastric glands accumulated [14C]AP indicating the existence of basal acid production in these glands, and EGF caused a small but significant reduction in basal [14C]AP uptake. A similar reduction of basal [14C]AP uptake was observed after exposure to omeprazole but not after ranitidine or prostaglandin E2 (PGE2). Histamine, dibutyryl cyclic AMP and K+ caused a strong and dose-dependent stimulation of acid formation by the glands. EGF, like omeprazole, reduced dose-dependently the [14C]AP accumulation stimulated by both histamine and dibutyryl cyclic AMP, while ranitidine and PGE2 reduced histamine- but not dibutyryl-cyclic-AMP-stimulated accumulation of [14C]AP. In the absence of other external stimuli, an increased K+ concentration enhanced [14C]AP accumulation to levels similar to those produced by histamine and this effect was not changed by EGF, ranitidine or PGE2 but was inhibited by omeprazole. We conclude that EGF interferes with the final steps of acid production between cyclic nucleotides and proton pump of the parietal cells.

Cyclic GMP and acid production in isolated gastric cells of the rat

Just as cAMP is regarded as an intracellular mediator of histamine, so has cGMP been connected with cholinergic stimulation of gastric acid secretion. The object of the present investigation was to study the possible role of cellular cGMP on 14C-aminopyrine uptake, an indirect measure of parietal cell H+-production, by using mixtures of isolated rat gastric cells and fractions with different parietal cell content. Cellular cAMP and cGMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced the cAMP and cGMP of gastric cells in a time- and concentration-dependent manner, by 98 and 124% (1 mmol/l) and was included in all further studies. In parietal cell enriched fractions, histamine elevated cAMP by 109% (100 mumol/l) without changing cGMP while carbachol did not influence either nucleotide. Various thiols and nitrogen compounds strongly enhanced cellular cGMP, e.g. hydroxylamine and L-cysteine (1 mmol/l) by 527 and 656%, whereas changes in cAMP were minimal. The hydroxylamine response occurred in parietal cell depleted and enriched fractions. 14C-aminopyrine (AP) uptake. IBMX alone reduced the basal AP uptake, potentiated the effect of histamine, and inhibited the effect of carbachol, which alone stimulated basal accumulation by 302%. The most efficacious stimulant of parietal cell H+-production was dibutyryl cAMP (582%, 100 mumol/l), whereas dibutyryl cGMP was without effect. However, this latter compound (1 mmol/l) reduced AP accumulation due to dibutyryl cAMP almost completely. Thiols and nitrogen compounds all more or less reduced AP uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate cyclase and H + production of isolated rat parietal cells in response to glucagon and histamine

The effect of glucagon and its interaction with histamine on adenylate cyclase (AC), cellular cAMP and [ 14C]aminopyrine ([ 14C]AP) uptake, a reliable index of parietal cell H + production, was studied in isolated rat gastric cells. AC activation in response to glucagon and histamine correlated with the number of parietal cells. Glucagon (10 −10-10 −6 mol/l) increasingly stimulated AC (maximal effect: 92% by 10 −7 mol/l) and cellular cAMP (86% by 10 −9 mol/l) of fractions enriched with 80% parietal cells but did not cause a pronounced change of the histamine-stimulated enzyme. If there was any interaction, the effect of both hormones was additive. Glucagon neither changed basal [ 14C]AP uptake nor interfered with that in response to histamine. The data suggest that if glucagon activates a parietal cell AC this process is not followed by parietal cell H + production. Furthermore, unlike other inhibitors such as somatostatin or PGE 2, glucagon does not reduce acid secretion via the cAMP system of the parietal cell.