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.     

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.     

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)     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha.     

Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.     

The effects of glycoprotein modification on beta-adrenoreceptors binding ability in rat cardiac membranes

The present study was designed to test the effects of neuraminidase and some proteolytic enzymes on the binding of 125I-HYP (hydroxybenzylpindolol) to beta-adrenoreceptors of rat cardiac membranes. The influence of some S-S and -SH active agents on the ligand binding used was also examined. The decrease in membrane sialic acid content did not alter the binding of ligand used. The degradation of membrane protein by proteases resulted in a time and enzyme type dependent decrease in the binding of 125I-HYP. It was found that the dithiotreitol pretreatment produced more profound decrease in ligand binding than reduced glutathione. Cysteine and mercaptoethanol were ineffective. The effect of dithiotreitol was dependent on the time of preincubation and on the agent concentration used. The decrease in ligand binding was also observed after the alkylation of -SH groups by iodoacetamide and N-ethylmaleimide.     

Action of modifiers of a radiation lesion on the binding of prostaglandin E2 with liver membranes in F1 CBA X C57BL strain mice

Prostaglandin E2 (PGE2) was specifically bound by the membrane fraction prepared from the mouse liver. The binding constants indicate the presence of high-affinity PGE2 binding sites with an apparent dissociation constant (Kd) of 0.82 X 10(-9) M and a capacity of 0.36 X 10(9) M/mg protein and a lower affinity PGE2 binding site with Kd = 15.73 X 10(-9) M and a capacity of 5.31 X 10(9) M/mg protein. The radioprotectors, MEA and APAETP inhibit PGE2 binding and alter its kinetics. Apparently the mechanism of PGE2 binding by membranes is related to interaction of prostaglandins with thiols and sufhydryl groups of membrane lipoproteins, while the radioprotectors modify the functional groups participating in receptor PGE2 binding.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Arginine and cysteine residues in the ligand binding site of alpha 2-adrenergic receptors

alpha 2-Adrenergic receptors were identified in calf brain, human platelet and human uterus membranes by [3H]-rauwolscine binding. The reagents phenylglyoxal (selective for guanidino groups), p- hydroxy mercuribenzoate and N-ethylmaleimide (selective for sulfhydryl groups) caused a time- and dose- dependent decrease in the number of receptor sites. alpha 2-Adrenergic agonists and antagonists mediated efficient protection of the receptors against these reagents. These data suggest that essential arginine and cysteine residues are present at or near the alpha 2-adrenergic binding site.     

Inhibition of adenylate cyclase by arsenite and cadmium: evidence for a vicinal dithiol requirement.

The effect of sodium arsenite and cadmium chloride on adenylate cyclase activity was examined in turkey erythrocyte membranes. Sodium arsenite was a weak inhibitor of adenylate cyclase -7mM produced only 60% inhibition. Its effect, however, was greatly potentiated by equimolar 2,3 dimercaprol- wherein 0.7 mM sodium arsenite inhibited 100% with an apparent Ki of 0.1 mM. Equimolar mercaptoethanol was less effective in potentiating sodium arsenite inhibition. Thus 0.7mM sodium arsenite in the presence of equimolar mercaptoethanol inhibited adenylate cyclase 56%. Excess 2,3 dimercaprol reversed inhibition by sodium arsenite or cadmium chloride. Sodium arsenite or cadmium chloride inhibited all forms of adenylate cyclase activity tested, including nonhormonal stimulation. Equimolar sodium arsenite and dimercaprol, at concentrations that caused 100% inhibition of adenylate cyclase activity, reduced the binding of the beta-receptor specific ligand iodohydroxybenzylpindolol by less than 15%. These results suggest that turkey erythrocyte membranes contain closely juxtaposed thiol groups and that interaction of such groups with arsenate interferes with the catalytic function of adenulate cyclase.     

Channel modulators affect PGE(2) binding to bovine aortic endothelial cells

PGE(2), PGF(2alpha) and the thromboxane agonist U-46619 bind to bovine aortic endothelial cells and compete on the same binding site with similar affinity. In addition, binding remains unaffected by prolonged exposure to the ligand. These characteristics differ significantly from those of any known G-coupled prostaglandin receptor. Binding of PGE(2) to the cells is reduced in the presence of the cyclic nucleotides cGMP and cAMP, and is unaffected by protein kinase inhibitors. Removal of permeable cyclic nucleotides from the cell medium results in a fast and complete restoration of PGE(2) binding to the cells, suggesting that both cyclic nucleotides reduce PGE(2) binding by a reversible interaction with the prostaglandin-binding site, without the involvement of second messenger-activated protein kinases. Our data further show that binding of prostaglandins to bovine aortic endothelial cells is sensitive to heavy metals and to activators and blockers of calcium, ATP-sensitive K(+) and chloride channels. Nickel, a specific cyclic nucleotide-gated (CNG) channel activator, decreases PGE(2) binding and so do the CNG channel activators Rp-8-Br-PET-cGMPS and Sp-8-Br-PET-cGMPS. On the other hand, the calcium channel blockers pimozide, diltiazem as well as LY-83,583, a guanylate cyclase inhibitor, which were reported to block CNG channels, enhance PGE(2) binding. The sensitivity of PGE(2) binding to selective CNG channel modifying agents, as well as the rapid and reversible interaction with cyclic nucleotides, may suggest that the common low-affinity prostanoid-binding site on bovine aortic endothelial cells is associated with a molecular entity, which possess several properties of a CNG channel.     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.     

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.     

Characterization of gastric mucosal prostaglandin E2 receptor

1. The binding characteristics of gastric mucosal prostaglandin (PG) E2 (PGE2) receptor were investigated using mucosal cell membranes from rat stomach. The binding was found to be dependent upon PGE2 and membrane protein concentration, the time of incubation and the pH of the mixture, being highest at pH 3.0. 2. Scatchard analysis of the binding data revealed a curvilinear plot with high affinity binding (Kd = 2 nM; Bmax = 0.106 pmol/mg protein) and low affinity binding (Kd = 319 nM; Bmax = 2.262 pmol/mg protein) sites. 3. Competitive displacement study indicated that the receptor was specific for PGs of the E series, as PGF2 alpha and 6-keto-PGF1 alpha failed to displace the PGE2. 4. The study is the first report to provide biochemical parameters of specific PGE receptors in rat gastric mucosa.     

Role of sulfhydryl groups in Y2 neuropeptide Y receptor binding activity.

Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity.     

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.     

Complex effects of sulfhydryl reagents on ligand interactions with nucleoside transporters: evidence for multiple populations of ENT1 transporters with differential sensitivities to N-ethylmaleimide

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate (pCMBS) were tested for their effects on ligand interactions with the [(3)H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no "reversal" in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.     

Association of a solubilized prostaglandin E2 receptor from renal medulla with a pertussis toxin-reactive guanine nucleotide regulatory protein

Prostaglandin E2 (PGE2) was found to bind specifically, reversibly, and in a protein-dependent manner to a single class of high affinity (KD approximately equal to 20 nM) binding sites in membranes prepared from canine renal outer medulla. PGE2 binding activity was solubilized from these membranes in a stable form (t1/2 greater than 14 days) in the absence of ligand in 75% yields using digitonin. The characteristics of PGE2 binding to membranes and solubilized protein were similar with respect to pH dependence, KD for PGE2, and order of potency of prostaglandins (PGE2 approximately PGE1 greater than PGF2 alpha greater than PGD2) in inhibiting the binding of [3H]PGE2. Importantly, the extents of binding of PGE2 to membranes and to a solubilized preparation partially purified by chromatography on wheat germ agglutinin-Affi-Gel 10 were both increased about 2-fold by GDP and GTP and its analogs. Treatment of the digitonin-solubilized PGE2 binding activity with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) rendered the binding activity insensitive to stimulation by GTP and decreased the apparent molecular weight of the peak of PGE2 binding activity from about 175,000 to about 65,000. These results suggest that the PGE2 binding activity resides in a protein which is tightly associated with, but distinct from, a guanine nucleotide regulatory (N) protein. PGE2 (greater than or equal to 10 nM) was found to stimulate GTPase activity of renal outer medullary membranes, and this stimulation was eliminated by pretreatment of membranes with pertussis toxin and NAD, but not cholera toxin and NAD. Treatment of both particulate and solubilized preparations of PGE2 binding activity with pertussis toxin plus NAD also eliminated the ability of GTP to stimulate PGE2 binding. This evidence indicates that it is the inhibitory guanine nucleotide regulatory protein, Ni, with which the PGE2 binding activity is associated. Thus, this PGE2 binding activity is an inhibitory PGE2 receptor, quite possibly one that mediates inhibition of vasopressin-induced cAMP formation in the medullary thick ascending limb and/or collecting tubule of the kidney.     

Effects of 16, 16-dimethyl prostaglandin E2 on bile-induced histamine release and permeability alterations in canine oxyntic mucosa.

Damage to the stomach results in excessive movement of hydrogen ion (H+) out of the lumen, and increased movement of sodium (Na+) and potassium (K+) into the lumen. Histamine liberation during damage probably adds to the destruction by increased capillary permeability and formation of edema. Previous reports have shown that the synthetic prostaglandin analogue 16,16-dimethyl prostaglandin E2 (Dm PGE2) protects dog gastric mucosa from aspirin- and ethanol-induced gastric mucosa damage. The effects of dm PGE2 on bile salt (sodium taurocholate) induced injury has not been investigated. Using the canine Heidenhain pouch, the present study examined the action of dm PGE2 on gastric mucosal damage induced by 5 mM sodium taurocholate in 100 mM HCl. Bile salt damaged the pouch mucosa as evidenced by an increased loss of H+, and increased net fluxes of both Na+ and K+. There was also an increase in the histamine content of the fluid irrigating the Heidenhain pouch. Intravenous injection of dm PGE2 in the doses 0.1 and 1.0 microgram/kg 1/2 h before administration of the sodium taurocholate in HCl significantly reduced the net loss of H+ and the gain of Na+, K+, and histamine. It is concluded the dm PGE2 effectively protects the canine gastric mucosa from the damaging effects of bile salt and that the mechanism of dm PGE2 protection of canine oxyntic mucosa may be mediated in part via inhibition of the gastric mucosal release of histamine.     

Chemical probing of the light-induced interaction between rhodopsin and G-protein. Near-infrared light-scattering and sulfhydryl modifications

Near-infrared light scattering and gel electrophoresis were used to monitor the interaction between rhodopsin and G-protein at photoreceptor disc membranes. It is found that substitution of one SH hydrogen at the G alpha-subunit by N-ethylmaleimide or thionitrobenzoate still allows dark binding of the G-unit to the membrane but blocks its light binding to rhodopsin. Repair of the modification restores the interaction of the proteins. Evidence is provided that substitution by the less bulky cyanide is also able to remove the hindrance to light binding. The rhodopsin-G interaction is reduced in proportion to bound N-ethylmaleimide, and the interaction kinetics remain constant over the measurable range. GTP/GDP exchange at the G-protein after interaction with rhodopsin does not reduce the accessibility of the relevant SH group. In contrast to the effect of G-protein modification, even exhaustive modification of rhodopsin has no effect on the dark and light binding of G-protein.