Evidence for nongastrin-mediated somatostatin inhibition of parietal cell function.

Somatostatin (SRIF), a tetradecapeptide, has been reported to suppress gastrin release and hence inhibit acid secretion in vivo. This study was performed to determine whether SRIF has any direct effect on parietal cell (PC). Isolated gastric cells were prepared by collagenase digestion and calcium chelation of rabbit fundic mucosa. PC enrichment (75% +/- 5%) was accomplished by velocity sedimentation with an elutriator rotor. Acid, as assessed by the accumulation of 14C-aminopyrine (AP) and macromolecular (intrinsic factor [IF]) secretion were used as markers of PC function. Cells were stimulated with histamine (H) (10(-6) mol/L). SRIF (10(-10) to 10(-6) mol/L) significantly inhibited H-stimulated 14C-AP accumulation (p less than 0.05). Inhibition of H-stimulated IF release was less sensitive, occurring at 10(-8) and 10(-7) mol/L (p less than 0.05), and loss of inhibition was observed at 10(-6) mol/L (p less than 0.05). These results demonstrate a direct inhibitory action of SRIF on PC secretion. The difference in inhibitory effect on IF and proton secretion is consistent with the postulation that SRIF may function at more than one site within the PC.

Evidence for dual modulation of pepsinogen secretion using isoproterenol, carbachol, CCK-8, forskolin, 8 bromo-cAMP, and A23187 probes.

The cellular mechanisms by which pepsinogen (PNG) secretion is controlled are not understood. The aim of this study was to explore whether modulation of PNG secretion is mediated by cAMP or calcium-calmodulin (C-C). PNG secretion in isolated rabbit gastric fundic glands (IGG) was tested, using agents believed to act via cAMP or C-C. IGG were stimulated for 30 minutes with histamine (H) 10(-5) M, isoproterenol (I) 10(-5) M, carbachol (C) 10(-5) M, cholecystokinin-octapeptide (CCK-8) 10(-7) M, forskolin (F) 10(-5) M, 8 bromo-cAMP (8B) 10(-3) M, and A23187 (A) 10(-6) M. PNG levels were determined by spectrophotometric assay of hemoglobin digestion products. PNG amounts secreted were (mean per cent above basal levels of total IGG PNG units +/- SEM): H, -0.02 +/- 0.30%; I, 3.5 +/- 0.9%; C, 5.1 +/- 2.2%; CCK-8, 5.3 +/- 1.5%; F, 10.6 +/- 3.8%; 8B, 13.8 +/- 4.5%; A, 2.1 +/- 1.1%. All secretagogues except H stimulated PNG release significantly above basal levels (p less than 0.05). A primary histaminergic mechanism for pepsinogen secretion is unlikely. Since two other adenylate cyclase activators, isoproterenol and forskolin and the 3':5'-cyclic adenosine monophosphate analog 8-bromo cAMP stimulated pepsinogen secretion, cAMP-dependence is probable. Since carbachol, CCK-8, and A23187, which are believed to act via calcium-calmodulin, also stimulated pepsinogen secretion, this system, too, presumably plays a substantial role. Thus the data support a dual 3':5'-cyclic adenosine monophosphate/calcium-calmodulin modulation of pepsinogen secretion.

A manganese-dependent protein kinase in gastric gland cytosol.

A protein kinase activity completely dependent on Mn2+ was studied in the cytosolic fraction from rabbit-isolated gastric glands. The manganese-dependent protein kinase (MNPK) activity phosphorylated major 33 kd (pp33) and minor 140 kd (pp140) endogenous proteins. The MNPK activity displayed a Kact for Mn2+ of 7.5 mmol/L. MNPK showed no preference for adenosine triphosphate or guanosine triphosphate with a Kact of 10 mumol/L for both. The kinase was differentiated from other known kinases since calmodulin, cyclic adenosine monophosphate, and phospholipids failed to stimulate pp33 phosphorylation. Furthermore, the protein kinase inhibitors trifluoperazine, the Walsh inhibitor protein, and heparin, as well as the phosphatase inhibitor p-nitrophenylphosphate failed to alter MNPK activity. The results indicate that novel MNPK activity is present in gastric gland cytosol. Elucidation of intracellular protein kinase activities may provide insights into the regulation of gastric secretory process.

Paradoxical effect of trifluoperazine, a calmodulin antagonist, on pepsinogen secretion.

Pepsinogen secretion (PS) is modulated at the intracellular level by both cAMP and calcium ion. Cholecystokinin octapeptide (CCK-8), a potent stimulus for PS, is believed to act through calcium. The most extensively studied pathway for calcium-mediated modulation involves the formation of calcium/calmodulin complexes, leading to activation of calmodulin. We have therefore examined the hypothesis that an inhibitor of calmodulin might inhibit PS stimulated by CCK-8. The phenothiazine derivative trifluoperazine (TFP) was chosen as a calmodulin antagonist. We measured in vitro secretion of pepsinogen by isolated gastric glands as a function of TFP concentration 10(-6) M-5 X 10(-4) M), in the presence and absence of a maximal concentration of CCK-8 (10(-7) M). Cellular viability was determined by measurement of release of the enzyme lactate dehydrogenase (LDH) into the medium. TFP did not significantly inhibit PS stimulation by CCK-8 at any concentration (P greater than 0.05). At 10(-4) M, TFP actually augmented PS stimulation by CCK-8 (P less than 0.05). TFP alone significantly stimulated PS (P less than 0.05) at 5 X 10(-5) M and above. TFP did not raise cAMP levels at any concentration tested (P less than 0.05), in contrast to the adenylate cyclase activator forskolin, 10(-5) M, which caused a 6- to 37-fold increase (P less than 0.05). TFP, 2 X 10(-4) did not increase LDH levels significantly (P less than 0.05). Thus a calmodulin inhibitor, TFP, paradoxically stimulates PS. This stimulatory effect of TFP is not cAMP-dependent and is not accompanied by a nonspecific release of LDH into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide augmentation of cholecystokinin-8-stimulated pepsinogen secretion: evidence for dual modulation of chief cell function.

Pepsinogen (PPG) secretion from chief cells (CC) is dually modulated by adenosine 3':5'-monophosphate (cAMP) and calcium second messenger systems. Vasoactive intestinal peptide (VIP) stimulates cellular function by elevating intracellular levels of cAMP. In contrast, cholecystokinin-8 (CCK-8) acts by producing a rise in intracellular calcium concentration. Consequently, it was the purpose of this study to test whether VIP (acting through cAMP-mediated systems) could augment CCK-8 (acting through calcium-dependent systems)-stimulated PPG secretion. Collagenase dispersed rabbit isolated gastric glands (IGG) were incubated alone (unstimulated) or with secretagogues for 30 min. VIP in graded doses of 10(-11) to 10(-7) M was used alone or in combination with CCK-8 (10(-9) M). PPG levels were determined using an assay based on pepsin hydrolysis of [14C]methemoglobin. Results are expressed as percentage of total pepsinogen (within the IGG) secreted above unstimulated levels. VIP alone (10(-11) to 10(-7)) or CCK-8 alone (10(-9)) did not significantly stimulate PPG secretion (P greater than 0.05). The combination of CCK-8 (10(-9) M) plus VIP (10(-7) M) significantly stimulated PPG secretion above unstimulated levels (P less than 0.05). Thus, the combination of VIP and CCK-8 produced greater PPG secretion than either secretagogue alone. These data support the hypothesis that secretagogues acting through either cAMP or calcium-mediated systems contribute to the regulation of PPG secretion from CC and that the two second messenger systems act in concert achieving at least additive effects.

Proton pump inhibition--the ultimate control of acid secretion?

The cellular mechanisms of acid secretion by the parietal cell (PC) include stimulation of membrane receptors, increases in cytosolic cyclic AMP levels, and activation of protein kinase systems. These events culminate in stimulation of a membrane-based proton pump. This consists of a non-electrogenic H+-K+-ATPase which transports H+ ions into the secretory canaliculus of the PC in exchange for the cation K+. It has been proposed that blockade of this proton pump would result in inhibition of acid secretion by all classes of acid secretagogues. Thus, the effects of membrane receptor agonists as well as any agents which augment cellular cAMP levels should be inhibited. Substituted benzimidazoles are weak bases which prevent acid secretion by blocking the H+-K+-ATPase system. In order to test the above hypothesis, we investigated the effects of the substituted benzimidazole H168/68 and cimetidine (C) on histamine (H) and 8B-stimulated acid secretion. The rabbit isolated gastric gland (IGG) model was used and acid secretion assessed by the accumulation of 14C-labeled weak base aminopyrine (AP) within the IGG in response to secretagogue stimulation. H168/68 and C both inhibited H (5 X 10(-5) M)-stimulated [14C]AP accumulation in a concentration-dependent manner (P less than 0.05). H168/68 inhibited both H- and 8B-stimulated [14C]AP accumulation (P less than 0.05), while C inhibited only H-stimulated [14C]AP accumulation (P less than 0.05). H168/68 suppressed [14C]AP below even unstimulated levels of [14C]AP accumulation. These results support the hypothesis that H168/68 inhibits the PC distal to cAMP stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Forskolin (cyclic adenosine monophosphate)-dependent protein phosphorylation in isolated gastric glands.

Improved management of peptic ulcer disease requires elucidation of cellular processes underlying gastric secretion. The intracellular execution of regulatory commands to secretory cells involves protein phosphorylation. We studied cyclic adenosine monophosphate (cAMP)-dependent phosphorylation in isolated gastric glands (IGGs) using forskolin, which directly stimulates adenylate cyclase. Forskolin stimulated secretion by both parietal and chief cells. In a separate set of studies, IGGs were incubated for 45, 90, and 105 minutes in modified Ham's F-10 medium containing orthophosphate labeled with phosphorus 32. The forskolin (10(-4) M) was added to some IGG preparations at 90 minutes. The reaction was terminated with sodium dodecyl sulfate and boiling. The proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, stained with Coomassie blue, and autoradiographed. Incorporation of phosphorus 32 increased progressively at 45, 90, and 105 minutes. Forskolin enhanced phosphorylated bands around 92 kilodaltons. These results are consistent with the major role of cAMP in the regulation of gastric cellular function. The study of cAMP-stimulated phosphorylation may be an important tool in the elucidation of intracellular regulatory mechanisms of gastric secretion. Modulation of these mechanisms may be the ideal therapeutic modality for treatment of acid-secretory disorders.

Reminiscence intervention in nursing homes: what and who changes?

The results of experimental studies of reminiscing are not as uniformly optimistic about its therapeutic value as is the theoretical literature. Moreover, anecdotal evidence regarding the impact of reminiscence interventions is more positive than the statistical evidence. This study suggests reasons for these previous findings, and examines data from a discussion group intervention in thirty nursing homes, in which 185 residents completed pre- and posttests. It analyzes the characteristics of participants in relation to modifications in selected attitudes and behaviors over the intervention period. Analysis of variance and discriminant analysis revealed no significant relationships between attitudinal or behavioral modifications and demographic or other characteristics, with one exception. Value-choices made by participants were related to such modifications. The findings are interpreted in terms of the "mental adaptability" of participants and the compensatory nature of reminiscence in an institutional setting.

The effects of cholecystokinin on parietal cell secretion in isolated gastric glands.

Cholecystokinin (CCK), a peptide hormone found in both gut and brain, shares amino acid homologies with gastrin and has previously been shown to stimulate gastric acid secretion in whole animal experiments. To investigate possible direct effects of CCK apart from extrinsic neural and hormonal influences, we have investigated the effects of the sulfated octapeptide of CCK (CCK-8) in rabbit isolated gastric glands using 14C-aminopyrine accumulation and intrinsic factor (IF) secretion as markers of parietal cell function. CCK-8 stimulated a significant increase (p less than 0.05) in IF secretion and 14C-aminopyrine accumulation. IF secretion was dose dependent for CCK concentrations from 10(-10)M to 10(-6)M. The combination of CCK (10(-6)M) and histamine (5 X 10(-5)M) elicited IF secretion greater than that of either agent alone. These results are similar to those observed for pentagastrin (10(-7)M), suggesting that the effects of CCK on parietal cell secretion may be due at least in part to a direct receptor-mediated parietal cell response to this agent.

Effects of ganglioside GM1 on the thermotropic behavior of cholera toxin B subunit.

The B, or binding, subunit of cholera enterotoxin forms a pentameric ring structure in the intact toxin, and also when the subunit is isolated from the A subunit. The thermal denaturation of the B subunit ring was examined by differential scanning calorimetry in the presence and absence of ganglioside GM1, its natural 'receptor'. In the absence of ganglioside an irreversible endotherm was observed with maximal excess apparent heat capacity, Cmax, at 74.6 degrees C. When the ganglioside was added in increasing amounts, multiple transitions were observed at higher temperatures, the most prominent having a Cmax at 90.8 degrees C. At high ganglioside concentrations, the 74.6 degrees C transition was not observed. In addition to the thermodynamic results a model is proposed for the interaction of GM1 and B subunit pentamer. This model is derived independently of the calorimetric results (but is consistent with such data) and is based upon considerations of the geometry of the GM1 micelle B subunit pentamer.

Assignment of the carbon-13 nuclear magnetic resonance spectra of gangliosides GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b.

Complete 13C nuclear magnetic resonance assignments are presented for gangliosides in the series GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. The gangliosides studied are related by the sequential addition of single saccharide residues. The structural relationships among these molecules were confirmed and subsequently utilized to provide the basis for a detailed investigation of 13C NMR oligomer-monomer shielding differences accompanying increasing oligosaccharide complexity. This gradual increase in complexity was reflected in the 13C NMR spectra and proved to be of significant value in the assignment task, resulting in the reassignment of four GM1 resonances from our previous work [Sillerud, L. O., Prestegard, J. H., Yu, R. K., Schafer, D. E., & Konigsberg, W. H. (1978) Biochemistry 17, 2619--2628]. The carboxyl-containing sialic acids in gangliosides have glycosidic linkage resonance shifts only approximately 30% as large as those found for neutral hexopyranosides; thus, care must be used in interpreting the 13C spectra of charge oligosaccharides. Secondary structural effects are also found to produce shifts in the resonances of the sialic acid adjacent to the GalNAc residue of GM2 and the more complex gangliosides, leading to inequivalence of the sialic acids in GD1a, GD1b, and GT1b.

Quantitative description of the binding of GM1 oligosaccharide by cholera enterotoxin.

We present a quantitative molecular interpretation of binding between the five B subunits of cholera enterotoxin and the oligosaccharide of ganglioside GM1, based on the currently accepted quaternary structure of the toxin and principles of multiple equilibria. A sequential binding equation is derived and fitted to published binding data obtained by equilibrium dialysis. In one study of binding to reduced toxin (I), intact toxin (II), and isolated B subunits (III) at low concentrations, analysis by the Hill equation suggested that binding was positively cooperative and that there were only four binding sites per toxin molecule; individual affinity constants could not be estimated because of the empirical nature of the Hill equation. Our analysis suggests that the evidence for positive cooperativity is stronger for I and III than for II. Affinity constants for the first binding step are about 2.0-2.1 microM-1 for I and 2.5-2.7 microM-1 for II and III; those for the second binding step are about 3.5-5.0 microM-1 and for I and III, but only 2.5 microM-1 for II. Constants for later binding steps are apparently within the range of 2-7 microM-1. Predictions of the sequential model at higher ligand concentrations diverge substantially from those of the Hill equation, and are supported by data obtained at higher protein and ligand concentrations. Thus all available equilibrium dialysis data are consistent with a single set of affinity constants and with the hypothesis of five equivalent binding sites.

Observation by 13C NMR of interactions between cholera toxin and the oligosaccharide of ganglioside GM1.

The initial event in the action of cholera toxin on intact cells is its recognition of cell-surface receptors, molecules of ganglioside GM1. We have studied details of this interaction by 13C NMR, which enables us to examine simultaneously both the protein and the ganglioside or, as in the present instance, its oligosaccharide portion. 13C NMR spectra of the toxin are consistent with the long correlation times expected for this 84,000-dalton protein. They show, however, some resolved resonances (including one tentatively assigned to the epsilon 2 carbon of tryptophan, 138.3 ppm downfield from tetramethylsilane). When oligosaccharide is added to the toxin this resonance broadens or moves further upfield to reside under phenylalanine resonances at 136.7 ppm. Of the seven tryptophan residues in cholera toxin, five are in the B subunits which bind GM1, so that the data are consistent with a resonance shift for the epsilon 2 carbons of these residues on binding the oligosaccharide. Resonances arising from the anomeric and methylene carbons of the sialic acid moiety of the oligosaccharide are also shifted. Comparison with corresponding chemical shifts in a series of model compounds suggest that the latter effects may originate in a toxin-induced conformational change in the oligosaccharide. At high resolution, anomeric carbon resonances of terminal galactose residues in free and bound oligosaccharide are also resolved.

Sequence homologies between A subunits of Escherichia coli and Vibrio cholerae enterotoxins.

The genes coding for the heat-labile enterotoxin LT produced by Escherichia coli have been cloned into the plasmid pBR313. Using DNA derived from the resulting chimeric plasmid, we determined the nucleotide sequence of two regions of the gene coding for the enzymatically active A subunit of LT. Translation of the nucleotide sequence gives the primary structure of the NH2-terminal and COOH-terminal regions of the LT A subunit. This permits direct comparison of the LT A subunit with the A subunit of cholera toxin. Our results show that the two toxins possess homologous sequences, of varying degrees, in both regions of their primary structure. The order of the component A1 and A2 polypeptides is A1-A2. The nucleotide sequence predicts the existence of a signal sequence of 18 amino acids at the NH2-terminus of the A subunit.

Evidence for involvement of cyclic nucleotides in intrinsic factor secretion by isolated rabbit gastric mucosa.

Secretion of intrinsic factor (IF) has previously been demonstrated in isolated rabbit fundic mucosa maintained in organ culture. We have now investigated the possibility that cyclic nucleotides may play a role in IF secretion. A phosphodiesterase inhibitor, 3-isobutyl methylxanthine (IBMX), stimulated IF secretion nearly fourfold while increasing tissue levels of both cyclic AMP (cAMP) and cyclic GMP (cGMP). Peak IF secretion in response to IBMX was not reached until after tissue cAMP levels were maximal. Dibutyryl cAMP and 8-Br-cAMP increased secretion by the same order of magnitude as did IBMX, whereas corresponding analogues of cGMP had no such effect. Histamine increased secretion of IF. In the presence of 40 microM IBMX, histamine elevated tissue levels of cAMP, but not of cGMP, and the stimulating effect of 10 microM histamine on IF secretion was potentiated. An H2 receptor antagonist, cimetidine, blocked the increases in IF secretion and tissue cAMP levels due to histamine, and the increase in IF secretion due to IBMX. These observations are consistent with a role for cAMP in the secretion of IF by isolated gastric mucosa.

Assignment of the 13C nuclear magnetic resonance spectrum of aqueous ganglioside GM1 micelles.

This article describes the natural-abundance Fourier-transform carbon-13 nuclear magnetic resonance spectrum, at 67.88 MHz, of aqueous micelles of bovine brain ganglioside GM1 of purity greater than 99%. Assignments are given for every carbon nucleus in the molecule, on the basis of a comprehensive study of the relevant mono-, di-, tri-, and polysaccharides, including several containing sialic acid (5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid), and phospho-, sphingo-, and glycosphingolipids. These assignments represent an extension of the 13C nuclear magnetic resonance data from monosaccharides and lipids to complex oligosaccharides and glycolipids. They also form the basis for interpretation of spectral perturbations induced in GM1 by titration with paramagnetic europium(III). The single sialic acid in GM1 was found to be alpha-glycosidically linked in the oligosaccharide from considerations of its unique anomeric chemical shift. The sialic acid carboxyl and glyceryl side chain, along with additional ligands donated by the 2-acetamido-2-deoxy-beta-D-galactopyranoside and terminal beta-D-galactopyranoside residues in the oligosaccharide portion of GM1, were found to be intimately involved with cation binding. It is proposed that the higher affinity, compared with monomeric sialic acid, of GM1 for cations may result from these additional oligosaccharide groups, which may effectively compete for water ligands in the metal cation coordination sphere.