The A2B adenosine receptor colocalizes with adenosine deaminase in resting parietal cells from gastric mucosa.

The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function.

Basolateral expression of GRP94 in parietal cells of gastric mucosa.

GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na2CO3 treatment dissociates part of it from the membrane. However, 100 mM Na2CO3 treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-ligand binding experiments. Both the K(D) value obtained in saturation experiments with N-ethylcarboxamido-[3H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.

Influence of alcohol consumption on immunological status: a review.

The aim of this review is to present and discuss the effect of different levels of alcohol consumption on the immune system. Not only the amount consumed but also the type of alcoholic beverage have to be taken into account in order to determine the consequences on activity, number, distribution, balance, interaction and response of immunocompetent cells. The association between alcohol exposure and the risk of developing an alcohol-related disease is multifactorial. In fact, age, gender, smoking habits, dietary intake and exercise are involved among other factors. The evaluation of the host cellular and humoral immune responses has shown that alcohol may induce some benefits when consumption is moderate. Moreover, those alcoholic beverages that contain antioxidants, such as red wine, could be protectors against immune cell damage. According to the literature consulted, the daily consumption of 10-12 g and 20-24 g of alcohol for women and men, respectively, is considered to be a moderate intake; the type of beverage has been established not to be important when defining moderation. Particular attention is often focused on the U- or J-shaped curve which also suggests that light to moderate drinking produces a protective effect. Such an inverse relationship indicates a reduction of risk for both light and moderate consumers and a higher risk not only for hard drinkers, but also for non-consumers.

P2Y purinoceptors in gastric gland plasma membranes.

This autoradiographic study of sections of the rabbit stomach fundus labelled with [35S]dATP alpha S, a radioligand for P2Y purinoceptors, has demonstrated a discrete pattern of distribution of the binding sites, i.e., the specific binding was only over the mucosa, but not over the muscular layer. Radioligand binding assays carried out on gastric gland plasma membranes showed that the binding process was saturable and a high density of a homogeneous population of binding sites was observed. These binding sites presented high affinity with a value of Kd = 4.1 +/- 0.8 nM and the maximum density of the binding sites was 16.8 +/- 1.6 pmol/mg protein. The displacement by purinoceptor ligands showed the following order of potency: ATP = 2-methylthio ATP > > alpha, beta-methylene ATP > > adenosine. Neither UTP nor pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were able to displace the binding. The data support the presence of P2Y purinceptors in rabbit gastric glands.

Binding of progesterone to specific sites in isolated hepatic cells and purified plasma membrane fraction.

Specific binding for progesterone has been determined in rat hepatocytes and mouse liver purified plasma membranes. The binding is saturable, reversible and temperature dependent. Two types of binding sites have been characterized in hepatocytes. The first is of high affinity and low binding capacity and the other one is of low affinity and high capacity of binding. In plasma membranes one type of specific binding site only exists. These high affinity sites are different from nuclear progesterone receptor, nuclear glucocorticoid receptor, digitalis receptor of Na+, K(+)-ATPase, transcortine and from corticoid binding sites determined previously in plasma membrane. We also have observed that specific progesterone binding to hepatocytes and plasma membrane is independent from the alpha and beta adrenergic receptors and from P-site adenosine receptor.

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes.

The specific binding of [3H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding are KD = 4.4 nM and Bmax = 685 fmol/mg protein in presence of 1 microM of corticosterone. With respect to the binding of 4 nM [3H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone greater than corticosterone greater than progesterone greater than cortisol greater than prednisolone greater than testosterone greater than 20 beta-hydroxyprogesterone greater than cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca2+ nor Mg2+ affected the binding of [3H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [3H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane.

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.

Characterization of cortisol binding sites in chicken liver plasma membrane.

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.