Assessing somatization in urologic chronic pelvic pain syndrome.

BACKGROUND: This study examined the prevalence of somatization disorder in Urological Chronic Pelvic Pain Syndrome (UCPPS) and the utility of two self-report symptom screening tools for assessment of somatization in patients with UCPPS. METHODS: The study sample included 65 patients with UCPPS who enrolled in the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Study at Washington University. Patients completed the PolySymptomatic PolySyndromic Questionnaire (PSPS-Q) (n = 64) and the Patient Health Questionnaire-15 Somatic Symptom Severity Scale (PHQ-15) (n = 50). Review of patient medical records found that only 47% (n = 30) contained sufficient documentation to assess Perley-Guze criteria for somatization disorder. RESULTS: Few (only 6.5%) of the UCPPS sample met Perley-Guze criteria for definite somatization disorder. Perley-Guze somatization disorder was predicted by definite PSPS-Q somatization with at least 75% sensitivity and specificity. Perley-Guze somatization disorder was predicted by severe (> 15) PHQ-15 threshold that had > 90% sensitivity and specificity but was met by only 16% of patients. The moderate (> 10) PHQ-15 threshold had higher sensitivity (100%) but lower specificity (52%) and was met by 52% of the sample. CONCLUSIONS: The PHQ-15 is brief, but it measures symptoms constituting only one dimension of somatization. The PSPS-Q uniquely captures two conceptual dimensions inherent in the definition of somatization disorder, both number of symptoms and symptom distribution across multiple organ systems, with relevance for UCPPS as a syndrome that is not just a collection of urological symptoms but a broader syndrome with symptoms extending beyond the urological system.

Production of ectopic gastric intrinsic factor in gastric mucosa of humans with chronic gastritis.

BACKGROUND: Ectopic expression of gastric intrinsic factor (IF) has been described in rodent models of chronic gastritis. AIMS: The current study undertook to determine if ectopic IF was also present in chronic gastritis in humans and might identify the process of ectopic protein expression as part of the response to chronic injury. METHODS: Archived biopsies from mid-body, angularis and prepylorus of 9 patients with and without chronic gastritis and food-cobalamin malabsorption were examined in a blinded fashion by immunocytochemistry as were biopsies from 5 normal subjects. Cells with ectopic IF were further examined with antibodies against pepsin or with Griffonia simplicifolia II (GSII) to identity cells in the mucous neck cell compartment. RESULTS: Ectopic IF production in non-parietal cells was identified in cells that were H(+),K(+)-ATPase-negative but IF-positive in 7 of the 9 patients (6/9 in the angularis and/or prepylorus biopsies and 1/9 only in the mid-body). These included 5 of the 6 H. pylori-infected patients and all 5 patients with severe food-cobalamin malabsorption. No normal control subjects demonstrated ectopic IF. The cells with ectopic IF were pepsinogen-positive peptic cells and were not GSII-positive. Expression was most extensive in patients and gastric regions with inflammation. In all but one sample, ectopic IF was observed near anatomical mucosal junctions, such as antral/body and prepylorus/duodenum junctions. CONCLUSIONS: These data in humans with and without gastritis are consistent with the hypothesis that local factors influence ectopic gastric IF expression, arising from either the anatomical location, the focal inflammation, or both.

Detection of apolipoprotein C in human and rat enterocytes.

Apoproteins have important physiologic functions in lipoprotein metabolism. Several apoproteins are produced in the intestine including ApoA-I, ApoA-IV, and ApoB. Each appears to participate in intestinal lipid transport. The liver also produces several apoproteins, including ApoC-II and ApoC-III, but the data demonstrating the ability of the intestine to produce ApoC is incomplete. Our aim was to ascertain whether ApoC-II and ApoC-III were present in human and rat jejunum, and if so, whether their presence was altered by fat feeding. The technique of immunolocalization and a newly developed double antibody radioimmunoassay for rat ApoC-III3 were used. ApoC-III3 was found in the supranuclear regions of enterocytes along the entire lengths of villi in the jejuna of 12-h-fasted rats. 1 hour after the gastric ingestion of corn oil. ApoC-III3 was found primarily in between cells and in the lamina propria. Similar results were obtained in human jejunal biopsies with ApoC-II and ApoC-III. ApoC-III3 was also detected by radioimmunoassay in enterocytes isolated from jejuna of neonatal and adult rats. Thus, ApoC-II and ApoC-III are clearly present in the intestine as well as in the liver. In addition, because their localization is altered after fat feeding, they are also likely to be produced in the enterocyte.

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation.

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.

Development of drugs for gastrointestinal motor disorders: translating science to clinical need.

Only a small number of new drugs have recently become available for gastrointestinal (GI) disorders. This is partly because we await outcomes of research into functional bowel disorder aetiology (e.g., role of microbiota) and of trials to control stress- related or painful GI symptoms (e.g., via CRF(1) receptors or beta(3) adrenoceptors). Nevertheless, only the ClC-2 channel activator lubiprostone has recently reached the clinic, joining the 5-HT(3) antagonist alosetron and the long-established 5-HT(4) agonist and D(2) antagonist metoclopramide; tegaserod, a non-selective ligand, was withdrawn. Interestingly, each has shortcomings, providing opportunities for molecules with 5-HT(4) or motilin receptor selectivity, and for new biology via guanylate cyclase C or ghrelin receptor activation. For translation into new drugs, the molecule must have appropriate efficacy, selectivity and pharmacodynamic properties. It is argued that the compound must then be evaluated in conditions where changes in motility are known to exist, before considering more difficult symptomatic conditions such as irritable bowel syndrome (IBS) or functional dyspepsia (FD), where relationships with disordered motility are unclear. Thus, it may be better to begin studying a gastric prokinetic in diabetics requiring improved glucose control, rather than in FD. Notably, new 5-HT(4) receptor agonists are being evaluated firstly as treatments of constipation, not IBS. New antidiarrhoeal agents should be developed similarly. Thus, progression of new drugs may require initial studies in smaller patient populations where clinical outcome is better defined. Only then can disease-related ideas be properly tested and drugs brought forward for these disorders (with high clinical need) and then, if successful for IBS and FD.

Protein synthesis in intestinal mucosa: the effect of route of administration of precursor amino acids.

All cells in the intestinal villus of the rat are capable of synthesizing protein from amino acid precursors (l-leucine). Moreover, polyribosomes from both crypts and villi are equally able to incorporate l-leucine into protein. Unlike other tissues, e.g. liver, there is no diurnal variation of protein synthesis in the intestine of the unfed rat, whether leucine is administered intraluminally or intravenously. The route of administration of precursor (l-leucine) is important in determining which part of the villus incorporates the label into protein. After intravenous administration, protein from cells near the villus-crypt junction is most heavily labeled, whereas after intraluminal administration protein from cells near the villus tip is most heavily labeled. The pattern of proteins most heavily labeled by radioactive precursor is different in the villus when compared with proteins from the crypt cells. Smaller molecular weight membrane-bound proteins are preferentially labeled in the crypt cells, whereas on the villus the pattern of labeling is more evenly distributed among the various proteins. Moreover, intraluminal leucine is utilized for protein synthesis to a greater extent than that in the blood, when the concentration in both compartments is similar. Thus, intraluminal and intravenous injections of labeled precursor are not equivalent. Both routes should be considered in data for experiments measuring intestinal protein synthesis.

The relation of size to the relative rates of degradation of intestinal brush border proteins.

Proteins associated with intestinal brush borders and their various fractions were solubilized with sodium dodecyl sulfate and beta-mercaptoethanol, and separated by electrophoresis on acrylamide gels containing sodium dodecyl sulfate. Brush borders contain at least 15 proteins or subunits, ranging in molecular weight from 19,000 to 270,000. The largest proteins (170-270,000 mol wt), including the disaccharidases, are removed from the brush borders by papain. Proteins belonging to the remaining membrane, including alkaline phosphatase, have an intermediate size (53-140,000 mol wt). The proteins corresponding to the filamentous "core" of the microvilli are the smallest (19-45,000). The relative rates of degradation of these proteins were studied by following the rate of decline of (14)C-labeled leucine activity in specific proteins, and by the double isotope technique of Schimke in which leucine-(14)C was given to intact rats intraluminally 10 hr before an intraluminal dose of leucine-(3)H. Heterogeneity of (3)H/(14)C ratios and thus of rates of turnover of brush border proteins was noted. In general, the largest proteins (including the disaccharidases), were turning over the fastest. Other membrane proteins (i.e. alkaline phosphatase) had an intermediate rate of degradation, and "core" proteins turned over slowly. Thus, there was a general correlation between relative degradation rate and size.

Intestinal apoproteins during fat absorption.

To compare the roles of apolipoprotein (Apo) A-I, B, and E (or arginine-rich apoprotein, ARP) in the intracellular production of intestinal chylomicrons (and/or VLDL), these apoproteins were localized in rat intestinal mucosa by the light microscope method of indirect immunofluorescence. In addition, tissue levels of ApoA-I and ApoB were measured during fat absorption by radioimmunoassay. Antisera were produced using ApoA-I isolated from rat plasma high density lipoprotein, and ApoB and ARP from plasma VLDL by column chromatography. The apoproteins yielded single bands on polyacrylamide disc gel electrophoresis in urea and in sodium dodecyl sulfate. Anti-apoprotein antisera were produced in rabbits. These antisera appeared to be monospecific on double-antibody immunoprecipitation of 125I-labeled apoproteins. In fasted animals granular staining of ApoA-I was noted in the supranuclear (Golgi) regions of epithelial cells in the top third of the villus. At 30 min, when fat droplets were seen in the supranuclear cytoplasm of the cells along the top two-thirds of the villus, intense ApoA-I staining surrounded droplets in the cytoplasm. At later times when epithelial cells and lamina propria both contained fat droplets, bright ApoA-I stain surrounded many droplets in the supranuclear cytoplasm of cells and in the lamina propria. Over the same period of time, tissue levels of ApoA-I rose 10-fold. The distribution and time-course of ApoB staining was nearly identical with that of ApoA-I. Concomitantly, tissue ApoB levels doubled. By contrast, in fasting rat intestine, staining of ARP was sparse, punctate, and confined to the lower quarter of the villus. After fat feeding, stained droplets were seen only in the lamina propria near the base of the villus even though abundant ARP was found in cells along most of this length of the villus. Stain was never seen to surround any droplets inside cells. Thus, ApoA-I and ApoB appeared to participate in the intracellular assemply of lipoproteins in gut, whereas ARP did not, although ARP was found within mucosal cells. Liver and intestine differed in their stainable contents of ApoA-I and ARP. Whereas intestine stained heavily for ApoA-I and lightly for ARP, liver stained heavily for ARP and lightly for ApoA-I. Both organs stained for ApoB. These findings suggest that there may be some quantitative "specialization" of the two organs which secrete lipoproteins.

Effect of secreted Bacteroides proteases on human intestinal brush border hydrolases.

Selected bacteroides species secreted various amounts of protease and glycosidase into their growth medium. Bacteroides vulgatus, distasonis, and ovatus secreted the most (31-60% of total). The secreted protease was similar in action to the protease within the organism, in that it had a broad pH optimum of 6-9, a K(m app.) for casein of 0.1 muM, and was inhibited by benzamidine, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate (DIFP), and by an elastase inhibitor, Ac(Ala)(3)AlaCH(2)Cl. Exposure of human brush border preparations to the secreted protease reduced maltase and sucrase activities; the reduction could be prevented by DIFP. In contrast, brush border alkaline phosphatase activity either did not change or increased after exposure to bacterial secretions. >90% inhibition of secreted glycosidase using EDTA and p-chloromercuribenzoic acid did not prevent the reduction of brush border maltase and sucrase activity, suggesting that glucosidases were not likely to be involved in the destruction of brush border enzymes. Moreover, the bacterial proteases caused only a small net release of active maltase or sucrase from the brush border. Most of the loss of activity was due to destruction of the enzyme. Proximal bowel fluid of three patients with overgrowth contained DIFP-inhibitable protease that destroyed sucrase in isolated brush borders. A Bacteroides species was isolated from each sample that secreted protease and destroyed brush border sucrase. We conclude that in bacterial overgrowth syndromes, brush border damage may occur from protease(s) secreted by Bacteroides.

Effect of proteolytic enzymes on the binding of cobalamin to R protein and intrinsic factor. In vitro evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

Cobalamin (Cbl; vitamin B(12)) malabsorption in pancreatic insufficiency can be partially corrected by bicarbonate and completely corrected by pancreatic proteases but the mechanisms involved are unknown. Because saliva contains enough R-type Cbl-binding protein (R protein) to bind all of the dietary and biliary Cbl, it is possible that R protein acts as an inhibitor of Cbl absorption and that pancreatic proteases are required to alter R protein and prevent such inhibition. To test this hypothesis we studied the ability of R protein and intrinsic factor (IF) to compete for Cbl binding and ability of pancreatic proteases to alter this competition. Human salivary R protein bound Cbl with affinities that were 50- and 3-fold higher than those of human IF at pH 2 and 8, respectively. Cbl bound to IF was transferred to an equal amount of R protein with t((1/2))'s of 2 and 90 min at pH 2 and 8, respectively, and within several hours respective ratios of R protein-Cbl/IF-Cbl of 50 and 2 were observed. Cbl bound to R protein was not transferred to IF at either pH 2 or 8. Incubation of R protein with pancreatic proteases at pH 8 led to a 150-fold decrease in its affinity for Cbl. Incubation of R protein-Cbl with pancreatic proteases led to complete transfer of Cbl to IF within 10 min. Gel filtration studies with R protein-[(57)Co]Cbl and (125)I-R protein showed that pancreatic proteases partially degraded R protein. Pancreatic proteases differed in their ability to effect these changes with trypsin > chymotrypsin > elastase. Pancreatic proteases did not alter IF in any of the parameters mentioned above. Pepsin failed to alter either R protein or IF. THESE STUDIES SUGGEST THE FOLLOWING: (a) that Cbl is bound almost exclusively to R protein in the acid milieu of the stomach, rather than to IF as has been assumed previously; (b) that Cbl remains bound to R protein in the slightly alkaline environment of the intestine until pancreatic proteases partially degrade R protein and enable Cbl to become bound exclusively to IF; and (c) that the primary defect in Cbl absorption in pancreatic insufficiency is a lack of pancreatic proteases and a failure to alter R protein and effect the transfer of Cbl to IF. These studies also suggest that the partial correction of Cbl malabsorption observed with bicarbonate is due to neutralization of gastric HCl, since at slightly alkaline, pH IF can partially compete with R protein for the initial binding and retention of Cbl.

A possible role for rat intestinal surfactant-like particles in transepithelial triacylglycerol transport.

To further examine whether surfactant-like particles (DeSchryver-Kecskemeti, K., R. Eliakim, S. Carroll, W. F. Stenson, M. A. Moxley, and D. H. Alpers. 1989. J. Clin. Invest. 84:1355-1361) were involved in the transepithelial transport of lipid, alkaline phosphatase activity and surfactant-like particle content were measured in apical mucosal scrapings, enterocytes, lamina propria, and serum after inhibition of chylomicron transport. Serum triacylglycerol levels were decreased 60-76% by Pluronic L-81, fenfluramine, and choline deficiency compared with fat-fed controls. 5 h after triacylglycerol feed, alkaline phosphatase activity in all three experimental groups was decreased compared with controls by 52-69% in mucosal scrapings and by 33-72% in serum. A parallel decline (60%) in alkaline phosphatase activity occurred in the lamina propria of Pluronic-treated animals. Total particle content (measured by an ELISA using antiserum against purified particle) after Pluronic treatment was decreased in mucosal scrapings, lamina propria, and serum by 16, 22, and 29% at 3 h and by 33, 40, and 8%, respectively, at 5 h after fat feeding. In contrast, particle content was increased in enterocytes by 29% 3 h and by 8% 5 h after fat feeding. By electron microscopy, enterocytes from Pluronic- and fenfluramine-treated animals exhibited a two- to threefold increase in large intracellular cytoplasmic lipid globules and the appearance of lamellae in apposition, with a marked decrease in the number of surfactant-like particles overlying the brush border. These changes, produced by inhibition of chylomicron transport, in the distribution of surfactant-like particles and particle-bound alkaline phosphatase are consistent with a role for these particles in transepithelial triacylglycerol transport across and out of the enterocyte.

Intestinal surfactant-like material. A novel secretory product of the rat enterocyte.

Surface-active phospholipid-containing particles are traditionally considered to be the product of type II pneumocytes. We now demonstrate membrane-bound lamellar cytoplasmic organelles in adult and suckling rat enterocytes that are densely reactive with phospholipid-staining reagents. These structures were seen in the basolateral space, within the intercellular junctions, and unraveling on the lumenal surface, and were more abundant after fat feeding. Light scrapings of intestinal mucosa and lumenal washings that contained these bodies, as evidenced by morphology and biochemical analysis, lowered surface tension in a pulsating bubble assay. Production by normal enterocytes of material with surfactant-like appearance and properties demonstrates that these structures are present in extrapulmonary epithelia, and extends the possible range of their function beyond gaseous exchange, e.g., solute exchange or lubrication on membrane surfaces.

Regulation of intestinal and hepatic apoprotein synthesis after chronic fat and cholesterol feeding.

Although diet influences levels of lipoproteins and their corresponding apoproteins, its effects on the molecular regulation of apoprotein synthesis are relatively unknown. Male Sprague-Dawley rats were fed an atherogenic diet containing cholesterol and propylthiouracil (PTU). Intestinal apo AI and AIV mRNA concentrations were decreased by the atherogenic diet, but apo AI and AIV synthesis was increased in vitro (organ explants) and in vivo (polysome runoff), consistent with regulation at the translational level. In contrast, hepatic apo E mRNA concentration and synthesis were increased after the atherogenic diet, consistent with pretranslational regulation. The response to cholesterol feeding for hepatic apo AI and E showed a third pattern of regulation, in which synthesis increased and mRNA content remained stable or fell, again suggesting translational control, but polysome runoff synthesis was unchanged. The apparent importance of translational regulation in the intestine is consistent with the necessity for the tissue to respond rapidly to changes in intraluminal content.

Correction of cobalamin malabsorption in pancreatic insufficiency with a cobalamin analogue that binds with high affinity to R protein but not to intrinsic factor. In vivo evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

In vitro studies indicate that [(57)Co]cobalamin (Cbl) is preferentially bound to salivary R protein as opposed to intrinsic factor (IF) and that [(57)Co]Cbl bound to R protein is not transferred to IF at either pH 2 or pH 8. Incubation of R protein-[(57)Co]Cbl with pancreatic proteases causes a partial degradation of the R protein moiety and a rapid transfer of [(57)Co]Cbl to IF. We have postulated that the etiology of Cbl malabsorption in pancreatic insufficiency is an inability to partially degrade R protein because of a lack of pancreatic proteases. We have tested this hypothesis by determining the ability of a nonradioactive Cbl analogue, bound with high affinity by R protein but not by IF, to correct the malabsorption of [(57)Co]Cbl in patients with pancreatic insufficiency.R protein bound the Cbl analogue known as cobinamide with affinities that were the same and only 14-fold lower than those for Cbl at pH 8 and pH 2, respectively. Cobinamide was bound by IF with affinities that were 600,000- and 10,000-fold lower than those for Cbl at pH 8 and 2, respectively. The addition of 125 pmol of nonradioactive cobinamide to 0.5 pmol of [(57)Co]Cbl before being added to 1 pmol of R protein and 1 pmol of IF, markedly inhibited the ability of R protein to compete with IF for binding the [(57)Co]Cbl. Similar results were obtained with freshly aspirated gastric juice. This change was essentially indistinguishable from that observed previously when R protein or R protein-[(57)Co]Cbl was incubated in vitro with trypsin. The oral administration of 100 nmol of nonradioactive cobinamide in Schilling tests was equivalent to trypsin in its ability to completely correct the malabsorption of 0.4 nmol of [(57)Co]Cbl in three patients with pancreatic insufficiency. The fact that both trypsin and nonradioactive cobinamide inhibit the ability of R protein to compete with IF for [(57)Co]Cbl binding in vitro, and correct the mal-absorption of [(57)Co]Cbl in patients with pancreatic insufficiency in vivo, supports our hypothesis that the primary defect in Cbl absorption in this disease is an inability to partially degrade R protein because of a lack of pancreatic proteases.

Characterization of ileal vitamin B12 Binding using homogeneous human and hog intrinsic factors.

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates. THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.

Effect of the NK(3) receptor antagonist, talnetant, on rectal sensory function and compliance in healthy humans.

Visceral hypersensitivity is important in the pathophysiology of irritable bowel syndrome and thus a target for modulation in drug development. Neurokinin (NK) receptors, including NK(3) receptors, are expressed in the motor and sensory systems of the digestive tract. The aim of this study was to compare the effects of two different doses (25 and 100 mg) of the NK(3) receptor antagonist, talnetant (SB223412) with placebo on rectal sensory function and compliance in healthy volunteers studied at two centres. Rectal barostat tests were performed on 102 healthy volunteers, randomized to receive either oral talnetant 25 or 100 mg or placebo over 14-17 days. Studies were performed on three occasions: day 1 immediately prior to 1st dose, day 1 4 h postdose, and after 14- to17-day therapy. Compliance, and pressure thresholds for first sensation, urgency, discomfort and pain were measured using ascending method of limits, and sensory intensity ratings for gas, urgency, discomfort and pain determined during four random phasic distensions (12, 24, 36 and 48 mmHg). Talnetant had no effect on rectal compliance, sensory thresholds or intensity ratings compared with placebo. In general, the results obtained at the two centres differed minimally, with intensity scores at one centre consistently somewhat lower. At the doses tested, talnetant has no effect on rectal compliance or distension-induced rectal sensation in healthy participants.

The possible role of pancreatic proteases in the turnover of intestinal brush border proteins.

1. Intestinal brush border enzymes have heterogeneous rates of turnover, the largest proteins having the fastest turnover. Since the membrane faces the intestinal lumen, the effects of pancreatic factors were examined in mediating this turnover. Surgical subtotal pancreatectomy was used as an experimental model to study the turnover of brush border proteins in the absence of most pancreatic secretions. 2. Subtotal (95%) pancreatectomy of rats was found to cause elevations by about 50% of total activity and specific activities of certain brush border enzymes (maltase, sucrase, lactase), but not of others (alkaline phosphatase, trehalase). Rats were judged to be functionally deficient in pancreatic proteolytic enzymes (a) by demonstration of vitamin B-12 malabsorption, which was corrected by trypsin, and (b) by the finding of only about 20% of proteolytic activity appearing in the lumen after a test meal when compared to control. 3. To measure protein turnover in vivo the method of double labelling was used, where [3H]- and [14C]valine were administered intraduodenally in sequence 10 h apart. With this technique, a high 3H/14C ratio is correlated with rapid turnover. Proteins with apparent molecular weights of about 200 000-270 000 were found to turn over more rapidly than smaller proteins. 3H/14C ranged from 4.7 to 6.2 in animals without pancreatic insufficiency. In the face of decreased pancreatic proteolysis, the 3H/14C ratio was 2.3-3.1, similar to that of proteins with a slow half life. 4. Estimates of relative synthetic rates of large brush border proteins were lower than normal in pancreatectomized animals, but were constant over the period of the labelling experiment. The high enzyme levels in the face of lower synthetic rates confirms that, at the new steady rate, degradation rates must be slower for large brush border proteins in pancreatic insufficiency. 5. In vitro, using purified brush borders, unfractionated pancreatic enzymes were found to remove sucrase, maltase and lactase, but not alkaline phosphatase and trehalase. The enzyme most potent in this respect was the pancreatic protease, elastase. Non-proteolytic enzymes (amylase, lipase, phospholipase A) were inactive in removing enzyme from the brush border. The addition of elastase to pancreatectomized animals in vivo restored the rapid turnover rate of large brush border proteins. 6. A model is thus proposed for the normal catabolism of some large intestinal brush border proteins. It is suggested that the surface of intestinal absorptive cells is being constantly remodelled, and that certain surface enzymes are in part removed from the membrane by the action of pancreatic proteases. A possible special role for elastase is suggested.

Labelling of intestinal brush border membrane proteins in vivo using diazotised[125I]iodosulfanilic acid.

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [125I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [125I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted by 27000 x g from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.

In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.

Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.

Rat intestinal alkaline phosphatase secretion into lumen and serum is coordinately regulated.

We have reported the presence of intestinal alkaline phosphatase on particles with surfactant-like properties within enterocytes, on the luminal surface (light mucosal scrapings) and in the lumen of adult fat-fed rat intestines ((1989) J. Clin. Invest. 84, 1355). To test the physiological role of these particles, we compared the effect on particle secretion of a known inducer of luminal and serum alkaline phosphatase secretion (fat), with the effect of pharmacological stimulators (cholecystokinin and bethanecol). Fat induced a 2-3-fold increase in membrane-free phosphatase activity in serum, and in particle-bound alkaline phosphatase activity in proximal luminal washings and light mucosal scrapings, reaching a peak in both compartments 7 h after a corn oil feed. Bethanecol given subcutaneously induced a quantitatively similar increase in serum alkaline phosphatase activity and in particle-bound phosphatase activity in proximal light mucosal scrapings, reaching a peak 7.5 min after injection. Cholecystokinin also had a 2-3-fold stimulatory effect, 30 min after injection, on particle-bound phosphatase activity in proximal intestinal light mucosal scrapings and distal intestinal luminal washings. The increase in alkaline phosphatase activity in serum samples reached a peak 60 min after cholecystokinin injection. Thus, three independent stimuli increase both luminal and serum appearance of intestinal alkaline phosphatase. These data support the earlier findings that intestinal alkaline phosphatase secretion into the lumen is mediated by a secreted particle, further show that secretion into serum and lumen is coordinately regulated, and are consistent with the hypothesis that the rise in serum alkaline phosphatase activity could be related to extracellular release of the enzyme from the particles.