Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways.

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) >> Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst(2(b)) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst(2(a)) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst(2(a)) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst(2(a)) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades.

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.

Immunohistochemical localization of the somatostatin SST2(A) receptor in the rat brain and spinal cord.

The neuropeptide somatostatin is widely distributed in the CNS and is believed to play a role as a neurotransmitter or a neuromodulator. Somatostatin mediates its actions by the binding of the peptide to high affinity membrane receptors. The genes for five somatostatin receptor types have been cloned recently and Northern blotting and in situ hybridization studies have shown that the transcripts of all five types are expressed in the CNS. Here we report the cellular distribution of somatostatin sst2(a) receptor protein in the adult rat CNS, using a polyclonal anti-peptide antibody directed against a portion of the C-terminal domain of the receptor. The specificity of the affinity-purified antibody was demonstrated by Western blotting and immunolabelling of cells transfected with a hemagglutinin epitope-tagged version of the sst2(a) receptor. Immunohistochemistry showed a distinct distribution of the receptor protein in the rat brain. Cells and processes were labelled in a number of areas, including the basolateral amygdala, the locus coeruleus, the endopiriform nucleus, the deep layers of the cerebral cortex, the subiculum, the claustrum, the habenula, the interpenduncular nucleus, the hippocampus and the central grey. In the spinal cord, the substantia gelatinosa showed strongly-labelled cell bodies and their processes. This study provides an improved understanding of the distribution of the sst2(a) receptor in rat brain.

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells.

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst5 receptors (CHOsst5), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2. In VSMC, SRIF (0.1 nM - 1 microM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC50 8.0-8.6) of the stimulated regeneration induced by submaximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml[-1]), platelet-derived growth factor-BB (PDGF, 5 ng ml[-1]) or endothelin-1 (ET-1, 100 nM). SRIF (pIC50 8.8) also inhibited bFGF-induced regeneration of CHOsst5 cells. 3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml[-1]) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM - 1 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg ml[-1]). 4. The sst5 receptor-selective agonist, L-362,855 (pIC50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst5 cells whilst the sst2 receptor-selective agonist, BIM-23027 (pIC50 6.8), was approximately 20 times weaker than SRIF. 5. The sst5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst5 cells (estimated pKB values 8.8 and 8.3, respectively). 6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml[-1]) for 20 h. 7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst5 receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive Gi/Go proteins.

Potent antagonism by BIM-23056 at the human recombinant somatostatin sst5 receptor.

We have investigated the effects of somatostatin (SRIF) and the linear octapeptide BIM-23056 on changes in intracellular calcium ion concentration ([Ca2+]i) and on the formation of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in CHO-K1 cells transfected with the human recombinant SRIF sst5 receptor. SRIF elicited concentration-dependent increases in [Ca2+]i, with a pEC50 of 7.02 +/- 0.06, while BIM-23056 (1 x 10(-7) M) behaved not as an agonist but as a potent, surmountable antagonist of these increases in [Ca2+]i. The SRIF concentration-effect curve for increases in [Ca2+]i was shifted rightward producing an estimated pKB for the antagonist of 8.0. BIM-23056 (1 x 10(-7) M) also significantly attenuated Ins(1,4,5)P3 increases due to SRIF, but had no effect on either basal or uridine 5'-triphosphate (UTP) (1 x 10(-4) M) stimulated increases in the levels of [Ca2+]i or Ins(1,4,5)P3.

Molecular cloning and functional characterization of a rat somatostatin sst2(b) receptor splice variant.

1. The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy-termini only. The murine sst2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization. 2. This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst2(a) receptor. 3. In CHO-K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC50 values of 8.3 (sst2(a) and 8.4 (sst2(b)). 4. SRIF (3 pM-3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst2(a) cells (pIC50 10.5) and CHO-sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5. Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx. 6. This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

Cloning and characterization of the epsilon and zeta isoforms of the 14-3-3 proteins.

Two prominent proteins (30 and 33 kD) in a purified preparation of the sheep pineal gland were studied. Amino acid analysis of tryptic peptides indicated that the 33-kD protein was the epsilon isoform of the 14-3-3 family of proteins, and that the 30-kD protein was the zeta isoform. The sheep pineal gland was found to have six other 14-3-3 isoforms in addition to the epsilon and zeta, suggesting that copurification of the epsilon and zeta forms may reflect the existence of homo- or heterodimers comprised of these isoforms. To characterize 14-3-3 proteins further in the pineal gland, the full sequence of the epsilon isoform and a partial sequence of the zeta isoform were cloned from a rat pineal cDNA library and are reported here. Tissue distribution studies using Western blot analysis revealed that rat pineal and retina have levels of 14-3-3 protein similar to those found in brain, and that relatively low levels occur in other tissues. This investigation also revealed the epsilon isoform was present at high levels in the rat pineal gland early in development and decreased steadily thereafter and that 30-kD isoforms exhibited the inverse developmental pattern.

Mucus glycoprotein gels. Role of glycoprotein polymeric structure and carbohydrate side-chains in gel-formation.

The structure of mucus glycoprotein gels from the pig gastrointestinal tract was investigated by mechanical spectroscopy. Gastric, duodenal, and colonic mucus had the same mechanical profile, characteristic of a viscoelastic gel. The gel structure collapsed on destruction of the polymeric structure of the component glycoprotein by reduction with 0.2M mercaptoethanol or after proteolysis with papain. The progressive weakening of mechanical properties and the decrease in polymeric glycoprotein content were measured as functions of time of reduction. A linear correlation was obtained between the gel quality [defined by tan delta, the ratio of the loss modulus (G'') to the storage modulus (G')] and the proportion of polymeric to subunit glycoprotein in the mucus. Purified mucus glycoprotein, at the same concentration as that in native mucus, resulted in a gel with mechanical properties no different from those of the respective native secretion, demonstrating that the glycoprotein alone could reproduce the gel-forming properties of mucus. After proteolytic digestion, all native secretions and reconstituted mucus showed an absence of Newtonian behaviour in the frequency dependence of dynamic viscosity at low frequencies. This provided evidence that the noncovalent interactions, characteristic of the native gel matrix, were still present after proteolytic digestion when the nonglycosylated protein core accessible to proteinases had been removed. These results were interpreted to show (a) a common mechanism for gel-formation in gastric, duodenal, and colonic mucus; (b) that the polymeric structure of mucus glycoproteins confers the three-dimensional structure necessary for formation of the gel network; and (c) that noncovalent interactions which arise between the glycoprotein molecules by relatively stable interdigitation of the carbohydrate side-chains are involved in formation of the gel network.

Mechanical characterization and properties of gastrointestinal mucus gel.

Mechanical spectroscopy has been used to study the structure of mucus gel taken from the surface of the pig gastrointestinal tract. Mucus from stomach, duodenum and colon was insoluble and its mechanical properties, characteristic of a weak viscoelastic gel, were unchanged in saline, acid (pH 2) and denaturants. Small intestinal mucus gel which was of poorer quality, was disrupted following exposure to acid and denaturants. Concentration of purified glycoprotein produced gels that had mechanical spectra with the same profiles as the respective native secretion except for reconstituted small intestinal mucus which was of better quality and similar to the other native and reconstituted gels. Reduction of S-S linkages or proteolysis of all mucus gels caused a collapse of structure to give profiles typical of a viscous solution. This collapse of gel structure was shown to result from a breakdown of the covalent polymeric structure of the component glycoproteins. A linear correlation for mucus gels was observed between gel quality (as defined by tan delta) and the ratio of polymeric glycoprotein to its degraded lower molecular weight subunit. Human gastric mucus from a histologically normal stomach also had the characteristics of a weak viscoelastic gel, although that from patients with peptic ulcer disease has a significantly reduced content of polymeric glycoprotein.

The gastric mucosal epithelial barrier: role of mucus and fibrin.

A continuous layer of insoluble mucus gel is adherent to the luminal surface of the gastric epithelium. The true thickness of the gel and its continuity can only be observed on unfixed sections of mucosa since histological fixatives cause dehydration and denaturation of mucus. The mucus:bicarbonate barrier can protect the undamaged epithelium from the endogenous luminal aggressors acid and pepsin but does not appear to offer much protection against exogenous damaging agents such as topical alcohol. Following acute ethanol injury, damaged epithelium is replaced by cells migrating from the gastric pits. In rat gastric mucosa this process of re-epithelialisation is protected by a gelatinous coat ten times thicker than the normal adherent mucus layer. Our studies now show this coat to be a fibrin gel with mucus and necrotic cells. Evidence suggests that the existing mucus layer can act as a template for the fibrinogen--fibrin conversion. These results demonstrate that a fibrin based gelatinous coat, quite distinct from the adherent mucus layer and with considerable protective potential, can be formed over the repairing damaged gastric mucosa.

Studies on gastrointestinal mucus.

Mucus is secreted throughout the gastrointestinal tract. The primary secretion is the water insoluble gel adherent to the mucosal surface but substantial amounts of mucus also occur in the lumen. The following are discussed: the functions of mucus; the structure and properties of mucus; study of the adherent mucus gel on the mucosal surface; the effects on mucus properties of proteolysis; thiol agents; bile salts; acid and hyperosmolar solutions. Much of our work on mucus has been with that from the stomach but where possible studies on the intestines and colon are discussed. Studies show that mucous secretions from the different regions of the gastrointestinal tract have similar rheological properties although the component glycoproteins differ in their detailed structure.

The role of mucus in the protection of the gastroduodenal mucosa.

There is good evidence that the adherent mucus plays an important role in the protection of gastroduodenal mucosa from the endogenous aggressors acid and pepsin. Adherent mucus provides a stable unstirred layer which supports surface neutralization of acid by mucosal bicarbonate output and acts as a permeability barrier to luminal pepsin. The adherent mucus layer is continuous. True thickness of the mucus layer and its continuity can only be observed on unfixed sections of mucosa, since histological fixatives and preparation for electron microscopy can cause dehydration and shrinkage of the mucus gel. The structure of adherent gastric mucus is deficient in patients with peptic ulcer disease because of decreased polymerization of the component glycoproteins. This impairment of the mucus barrier is associated with raised amounts of pepsin 1, which digests the mucus layer more aggressively than the major pepsin, pepsin 3, under conditions that pertain both in the stomach (pH 2) and duodenum (pH 4-5). Adherent mucus does not appear to offer much protection against exogenous damaging agents, e.g. alcohol and aspirin. These agents permeate the mucus barrier, damaging the underlying epithelium. The subsequent epithelial repair process is protected by a gelatinous coat over ten times thicker and distinct from the normal adherent mucus layer. Our recent studies show this gelatinous coat to be primarily a fibrin-based gel with mucus and necrotic cells.

Prolonged activation of extracellular signal-regulated kinase by a protein kinase C-dependent and N17Ras-insensitive mechanism mediates the proliferative response of G(i/o)-coupled somatostatin sst(4) receptors.

The human sst(4) receptor, recombinantly expressed in Chinese hamster ovary cells, mediates proliferative activity of the peptide hormone somatostatin. This effect was shown to involve activation of pertussis toxin-sensitive G proteins and was inhibited by overexpression of the betagamma-sequestrant, transducin. Somatostatin-induced proliferation was abolished by the MEK1 inhibitor, PD 98059, whereas the Src inhibitor, PP1, had no effect. A marked increase was observed in the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) 10 min after sst(4) receptor activation, which was blocked by pertussis toxin, decreased by PP1 and the betagamma-sequestrant, but unaffected by PD 98059. In contrast, the somatostatin-induced phosphorylation of ERK obtained at 4 h, although sensitive to both pertussis toxin and transducin, was unaffected by PP1 but ablated by PD 98059. Protein kinase C inhibition also abolished this somatostatin-induced sustained phosphorylation of ERK, together with the associated increase in cell proliferation. Expression of dominant negative Ras (N17) failed to significantly reduce the proliferative effect mediated by the sst(4) receptor but markedly attenuated the acute phase of the somatostatin-induced phosphorylation of ERK obtained at 10 min. In contrast, the phosphorylation induced at 4 h was unaffected. We conclude that ERK activation by G(i/o)-coupled sst(4) receptors involves a Src and Ras-dependent acute phase, but the proliferative response is dependent upon the prolonged ERK-induced activity, mediated by protein kinase C.

Gastrointestinal mucus gel rheology.

Mucus secretions from human and pig stomach; pig duodenum and pig colon have the same viscoelastic gel structure, as determined by mechanical spectroscopy. These mucus gels are readily solubilised by proteolysis or reduction with thiol agents but are stable with an unchanged mechanical spectra following exposure to pH 1-8, bile, bile salts and hypertonic 2 M NaCl. Gels of the same mechanical spectra and stability are reproduced by concentration of the isolated mucins purified free of protein, DNA and lipid (less than 1%). A direct correlation is observed between percentage of total mucin in polymeric form in the mucus secretion and its gel quality. The mechanical spectra of proteolytically digested mucus, together with the resistance of mucus to denaturing agents suggest carbohydrate-carbohydrate interactions are involved in gel matrix formation. Pig submaxillary mucin forms model gels with the same viscoelastic gel structure as other gastrointestinal gels. Wide variations in the composition and length of the carbohydrate chains of mucins do not affect the gel-forming properties.

Formation of a fibrin based gelatinous coat over repairing rat gastric epithelium after acute ethanol damage: interaction with adherent mucus.

A gelatinous coat, heterogeneous in appearance, was formed over damaged rat gastric mucosa recovering from acute ethanol injury. This coat, in places 1.6 mm thick (median thickness 680 microns), was 10 times thicker than the translucent layer of adherent mucus (median thickness 70 microns) covering the undamaged mucosa. Immunohistochemistry and periodic acid Schiff staining showed this gelatinous coat to be predominantly a fibrin gel with an exterior layer rich in mucus and necrotic cells. The plasma clotting time was significantly decreased in vitro by pig gastric mucus gel and soluble mucus glycoprotein (90% and 13% respectively) suggesting that in vivo the mucus layer remaining after epithelial damage could act as a template for fibrinogen-fibrin conversion. These results show that a fibrin based gelatinous coat, quite distinct from the adherent mucus layer and with considerable protective potential could be formed over the repairing rat gastric mucosa after acute ethanol damage.

The mucus barrier. Its role in gastroduodenal mucosal protection.

A layer of water-insoluble mucus gel has been shown to form a continuous cover over the gastroduodenal mucosal surfaces, of median thickness of 180 micron in stomach in humans. This adherent mucus is the first line in mucosal defence against the natural aggressors, acid and pepsin, in the lumen. Mucus gel provides a stable unstirred layer that supports surface neutralisation of acid by mucosal bicarbonate. Mucus gel is a diffusion barrier to pepsin in the lumen, preventing proteolysis of the underlying epithelial cells. There is, however, a dynamic balance between digestion by pepsin of the mucus layer at its luminal aspect and secretion of new mucus by the epithelium. There is evidence that, in peptic ulcer disease, the rate of peptic degradation of the mucus barrier is increased. Exogenous damaging agents such as ethanol and aspirin permeate the gel matrix of the mucus barrier, rapidly damaging the underlying epithelium. The subsequent reepithelialisation process is protected by a gelatinous coat over ten times thicker than the original adherent mucus layer. This gelatinous coat is primarily a fibrin-based gel with necrotic cells and mucus.

High-intensity p38 kinase activity is critical for p21(cip1) induction and the antiproliferative function of G(i) protein-coupled receptors.

G protein-coupled receptors can stimulate the p38 kinase cascade, but the effect this has on cell growth remains poorly characterized. Here we show human somatostatin sst(2) and sst(4) receptors inhibit basic fibroblast growth factor (bFGF)-induced proliferation, via a mechanism that was blocked by the p38 inhibitor PD 169316. The sst(4) receptor could also induce a proliferative activity in the absence of bFGF, which was unaffected by PD 169316. In contrast, the sst(3) receptor had no effect on basal cell growth or on the proliferation evoked by bFGF. The extracellular signal-regulated kinase activity stimulated by the sst(3) receptor was transient in duration compared with a sustained activity induced by the sst(2) and sst(4) receptors and which was critical for the proliferative response of the latter receptor. In addition, activated sst(2) and sst(4) but not sst(3) receptors evoked a prolonged phosphorylation of p38 that was amplified by bFGF. The accumulation of the cell cycle inhibitor p21(cip1) was only apparent after sst(2) and sst(4) receptor activation in the presence of bFGF, which was sensitive to PD 169316 or pertussis toxin. Thus, the contrasting antiproliferative effects evoked by the human sst(2), sst(3), and sst(4) receptors can be accounted for by their differential abilities to activate p38. This activity is critical for p21(cip1) induction, blockade of entry into S phase, as indicated by the lack of retinoblastoma protein phosphorylation, and the associated antiproliferative activity of somatostatin. Furthermore, by changing the intracellular signaling threshold of p38 through cooperative effects of somatostatin and bFGF, the sst(4) receptor can mediate opposing effects on cell proliferation.

Somatostatin activates two types of inwardly rectifying K+ channels in MIN-6 cells.

Western blotting revealed the presence of five somatostatin receptor types, sst1, sst2, sst3, sst4 and sst5, in the mouse pancreatic -cell line MIN-6. In MIN-6 cells, glucose-induced electrical activity was potently (pEC50 = 12.7) and irreversibly reduced by somatostatin (SRIF-14); this was associated with hyperpolarization of the membrane potential (pEC50 = 11.2) and a decrease in the input resistance (pEC50 = 12.7). The effects of SRIF-14 were mimicked by 100 nM L-362,855 (a partial agonist at sst5 receptors), but not BIM-23027 or NNC-26,9100 (selective agonists at sst2 and sst4 receptors, respectively). CH-275 at 100 nM (a selective agonist at sst1 receptors) partially inhibited electrical activity but without membrane potential hyperpolarization. One hundred nanomolar SRIF-28 activated an inwardly rectifying K+ current (ISRIF) ISRIF was activated neither by 1 M BIM-23056 nor CYN-154806 (antagonists at sst5 and sst2 receptors, respectively). The activation of ISRIF by 100 nM SRIF-28 was, however, inhibited 93 % by BIM-23056; CYN-154806 had no effect. Both 100 nM glibenclamide and 200 M tolbutamide, blockers of the -cell ATP-sensitive K+ channel (K-ATP), reduced ISRIF by ~44 %, whereas 1 mM Ba2+ abolished ISRIF. In cell-attached patches, 100 nM SRIF-14 activated two types of single-channel currents whose properties were consistent with those of K-ATP and GIRK channels. In conclusion, somatostatin can inhibit glucose-induced electrical activity in MIN-6 cells by the combined activation of K-ATP and GIRK channels. Studies with selective agonists and antagonists are consistent with this effect being mediated by the sst5 receptor.

Misoprostol-induced increases in adherent gastric mucus thickness and luminal mucus output.

Gastroduodenal mucus can be separated into two phases: insoluble mucus gel adherent to the mucosal surface, and luminal mucus, which is removed by washing out the lumen. The adherent mucus gel is part of the mucosal protective barrier to acid and pepsin in the gastric juice. Luminal mucus, which is mobile, probably does not significantly protect against gastric juice, but functions as a lubricant, protecting the adherent mucus layer and underlying mucosa from mechanical damage. Adherent mucus is observed on the mucosal surface as a thin, continuous, gelatinous layer of variable thickness, about 50-450 microns (median, 180 microns) in man and 10-230 microns (median 80 microns) in the rat. Thickness of this adherent mucus layer in the rat stomach is increased significantly (up to threefold) following topical administration of misoprostol in vivo 1 hr before measurement. Simultaneous increases are observed in the content of luminal mucus following misoprostol administration. Seventy percent of maximum response is observed within 5 min of topical prostaglandin administration, compatible with the release of preformed mucus. Such prostaglandin-stimulated increases in mucus thickness will improve the protective capacity of the adherent mucus gel. The thickness of the adherent mucus layer is not changed following topical exposure, in vivo 1 hr before measurement, to exogenous mucosal-damaging agents (eg, ethanol, indomethacin and taurocholate. However, since such damaging agents permeate the mucus gel, it appears to offer little initial protection to the underlying epithelium. The mucus barrier primarily guards against the natural aggressors acid and pepsin, protecting the epithelium and its repair following acute mucosal damage.

Submaxillary mucins. Intermolecular interactions and gel-forming potential of concentrated solutions.

The intermolecular interactions in concentrated solutions of pig submaxillary mucin (PSM) and sheep submaxillary mucin (SSM) were studied by mechanical spectroscopy. PSM and SSM were purified from detectable protein and nucleic acid by equilibrium centrifugation in a CsCl density gradient. PSM and SSM isolated in the presence of proteinase inhibitors showed distinct differences from preparations isolated in the presence of 0.2 M-NaCl alone, the latter having a carbohydrate and amino acid analysis similar to other preparations isolated by precipitation or ion-exchange techniques. Gel-filtration studies showed that preparations isolated in the presence of 0.2 M-NaCl alone were dissociated into smaller-sized glycoprotein units by 3.5 M-CsCl or 2.0 M-NaCl (SSM), pH 2.0 (PSM) or heating at 100 degrees C for 10 min (PSM and SSM). Preparations isolated in the presence of proteinase inhibitors were not dissociated by these treatments. Proteolysis fragmented all submaxillary mucin preparations into small glycopeptides of Mr 13,700 for PSM and of Mr 14,000 and 15,000 for SSM. PSM preparations when concentrated formed viscoelastic gels, as determined by mechanical spectroscopy. In contrast, SSM showed characteristics of a weak viscoelastic liquid under comparable conditions (coil overlap). PSM glycoprotein isolated in proteinase inhibitors formed weak viscoelastic gels at concentrations between 5 and 15 mg/ml. Preparations of PSM glycoprotein isolated in the presence of 0.2 M-NaCl (concentration 10-97 mg/ml) had the same overall mechanical gel structure as those preparations extracted in the presence of proteinase inhibitors. This gel structure was seen to collapse following proteolysis of both preparations or after acid treatment of the glycoprotein isolated in the presence of 0.2 M-NaCl, consistent with the breakdown in size of the polymeric glycoprotein. Treatment of PSM gel with 0.2 M-2-mercaptoethanol caused a surprising increase in gel strength, which was further markedly increased on removal of the reducing agent by dialysis. An association of reduced subunits of PSM was observed by gel filtration after removal of 0.2 M-2-mercaptoethanol. These results point to intermolecular disulphide exchange occurring on reduction of these PSM glycoprotein preparations. These results demonstrate that gel formation in PSM glycoprotein is similar to that for other gastrointestinal mucus glycoproteins from stomach to colon. Gel formation in PSM, as in other mucins, depends on polymerization of subunits.(ABSTRACT TRUNCATED AT 400 WORDS)