Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE2 binding sites in four subcellular fractions (F1-F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of 3HPGE2 to fractions F2-F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitrochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE2 binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Effects of enzymes and protein modifying reagents on the binding of 3H-prostaglandin E2 to porcine oxyntic mucosa invitro

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE₂ binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A₂ was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinzation of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and β-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE₂ and its binding site. The ability of mercaptoethanol and N-methylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE₂ binding reaction.     

Effects of enzymes and protein modifying reagents on the binding of 3H-prostaglandin E2 to porcine oxyntic mucosa in vitro

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE2 binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A2 was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinization of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and beta-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE2 and its binding site. The ability of mercaptoethanol and N-ethylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE2 binding reaction.     

Postnatal development of somatostatin levels and its binding sites in rabbit gastric mucosa

Using a specific radioimmunoassay technique, we have determined somatostatin-like immunoreactivity (SLI) in acid extracts of gastric (fundic and antral) mucosa as well as the specific binding of ¹²⁵I-Tyr¹¹-somatostatin to cytosol of the stomach of 0 to 150 days postnatal rabbits. The levels of somatostatin in both fundus and antrum decreased from birth up to day 5 followed by a sharp increase from 5 to 10 days, then decreased progressively until day 35. After this age, the somatostatin concentration remained relatively stable. The number of specific somatostatin binding sites of both high- and low-affinity increased gradually (without changes in the affinity values) with the development of rabbits, reaching the adult level by 35 days. However, there was an apparent lack of high-affinity sites immediately after birth (day 0). The somatostatin binding sites had characteristics identical with those found in adult animals with regard to their respective specific ligands.     

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.     

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 X g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.     

Localization of ClC-2 Cl- channels in rabbit gastric mucosa

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl- channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl- channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.     

In vitro electrophysiological effects of cimetidine, prostaglandin E2 and acetylcholine on the rabbit gastric mucosa

The electrophysiological effects of cimetidine, cytoprotective dose of prostaglandin E2 (PGE2) and acetylcholine were determined in parallel in Ussing-chambered rabbit fundic and antral mucosal preparations. In the fundic mucosal preparations both cimetidine and PGE2 caused an increase in transmucosal potential difference (PD) and in short-circuit current (ISC); the transepithelial resistance (Rt) was essentially unchanged. Addition of acetylcholine to the pretreated fundic preparations produced further gradual increases in PD and ISC; cimetidine pretreatment delayed this effect of acetylcholine. In contrast to fundic mucosa, cimetidine did not cause any electrical change of the antral preparation but decreases in PD, Rt and ISC were detected after the addition of PGE2. Acetylcholine produced a rapid initial PD elevation followed by a PD drop of both antral tissues independent of pretreatment. These findings suggest that both cimetidine and PGE2 generated electrical hyperpolarisation of rabbit fundic mucosa. These changes may be favourable for mucosal protection. No "beneficial" electrical changes were detected on the antral mucosa after administration of cimetidine and PGE2. Acetylcholine increased the effects of other stimuli on the fundic mucosa. In the rabbit antral mucosa acetylcholine generated biphasic changes of electrical properties.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Somatostatin binding sites in cytosolic fraction of rabbit intestinal mucosa: distribution throughout the intestinal tract

Specific binding sites for somatostatin have been identified in cytosolic fraction of both small and large intestinal mucosa. The stoichiometric data suggested the presence of two classes of binding sites in each part of the intestine. The binding capacity varied depending on the segment considered (rectum greater than duodenum = jejunum greater than ileum, caecum and colon). However, the affinities of the binding sites were similar throughout the whole intestinal mucosa, with the exception of rectum which showed higher Kd values. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as vasoactive intestinal peptide, neurotensin, substance P and Leu-enkephalin did not show any effect upon somatostatin binding.     

Subcellular distribution of [H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa

Specific binding sites for somatostatin have been found in the cytosolic fractions of both parietal and non-parietal cells from rabbit gastric fundic mucosa. The stoichiometric data suggested the presence of two classes of binding sites in both types of ceils. The number of low-affinity binding sites was significantly higher in parietal cells than in non-parietal cells. The reverse was true for the high-affinity binding sites. However, the affinity of each class of binding sites was similar in the cytosolic fractions of both parietal and non-parietal ceils. It thus appears that low-affinity somatostatin binding sites are mainly located in the parietal ceils whereas the high-affinity sites occur principally in the non-parietal cells.     

Effects of enzymes and protein modifying reagents on the binding of H-prostaglandin E2 to porcine oxyntic mucosa

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE₂ binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A₂ was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinzation of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and β-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE₂ and its binding site. The ability of mercaptoethanol and N-methylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE₂ binding reaction.     

Subcellular localization of prostaglandin E2 receptors in the gastric mucosa.

Gastric mucosal PG E2 receptors are the common antisecretory working point of all prostanoid types and may also be involved in "protective" effects. We investigated the subcellular localization of these receptors, as measured by displaceable 3H-PG E2 binding, and identified different organelles by monitoring the activities of specific marker enzymes. Porcine mucosal homogenates were subdivided by differential centrifugation into fractions P1 (1000 x g), P2 (20,000 x g), P3 (300,000 x g) and the supernatant S1. P3 was further fractionated over a series of sucrose step gradients. Mitochondria and lysosomes were enriched in P2 (maximum specific activities of cytochrome-c-oxidase of beta-glucosidase, beta-glucuronidase, beta-galactosidase, respectively). Plasma membranes (alkaline phosphatase, gamma-glutamyl-transpeptidase, 5-nucleotidase), tubulovesicles (H+/K(+)-ATPase) and rough endoplasmic reticulum (NADPH-cytochrome-c-reductase) were mainly found in P3, which also contained the majority of 3H-PG E2 binding sites. In contrast, prostanoid binding was barely detectable in S1. Density fractionation of P3 revealed that 3H-PG E2 binding sites shared a similar sedimentation profile with plasma membranes and tubulovesicular markers. No or negative correlation was found with lysosomes, rough endoplasmic reticulum and mitochondria. We conclude that mucosal PG E2 receptors are predominantly located at the cell surface. This supports the view that prostanoids inhibit gastric secretion through membrane receptors, but gives no clue for intracellular "protective" working points.     

Effect of small intestinal resection on somatostatin binding to cytosol of rabbit gastric mucosa

Small bowel resection in the rabbit increased gastric (fundus and antrum) somatostatin content and decreased the number of somatostatin binding sites (but not their corresponding affinity values) in gastric (fundus and antrum) cytosol three weeks after surgery. Five weeks after resection the number of somatostatin binding sites at both fundic and antral levels as well as antral somatostatin content returned towards control values whereas the fundic concentration of the peptide remained increased. Present results together with the known inhibitory role of somatostatin on various gastric functions suggest that the gastric alterations showed by animals subjected to small bowel resection may be due, at least in part, to the observed decrease of the number of gastric somatostatin binding sites.     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Biogenic amine binding sites in rabbit spermatozoa

The binding of the neuroleptic, [3H] spiperone, to rabbit spermatozoa was investigated. Binding was partially inhibited by haloperidol, mianserin or epinephrine. Mixtures of two of the three inhibitors reduced binding further, but binding of the radioligand to spermatozoa was lowest in the presence of all three inhibitors. Unlabeled spiperone eliminated binding. Cholinergic ligands, GABA, and beta-adrenergic ligands, had little effect on binding. Spiperone binding occurred to both heads and tails suggesting that alpha 1-adrenergic, D2-dopamine and S2-serotonin binding sites are present in the head and/or tail of rabbit spermatozoa.     

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatinlike immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr¹¹-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific, somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.