Distribution of alkaline sphingomyelinase activity in human beings and animals

The alkaline sphingomyelinase (SMase) was first found in rat intestinal brush border. The important roles of this enzyme in digestion of sphingomyelin and in mucosal cell proliferation have been suggested. In the present work, the distribution of the alkaline SMase in the tissues of human beings and animals have been studied. By assaying the enzyme activity in human biopsy samples, we found that the alkaline SMase activity was absent in the stomach, increased in the duodenum, present at high levels in the small intestine, and slightly declined in the colon and rectum. High activities were found similarly in the intestinal contents of the healthy adults and infants. The activities were also found in the intestinal mucosa of rats, normal and germ-free mice, and hamsters with the same distribution pattern as in humans, but not in the intestinal mucosa of guinea pigs. Apart from the intestinal tract, a SMase activity preferring alkaline pH was identified in human and guinea pig bile, but not in the bile of rat, pig, sheep, and cow. No activity was found in either pancreatic tissue or pancreatic juice in all species tested, and none was detected in human urine and milk. In conclusion, alkaline SMase exists predominantly in the digestive system with considerable tissue and species differences.     

Distribution of 3α-hydroxysteroid dehydrogenase (bile acid binder) in rat small intestine: Comparison with glutathione S-transferase subunits

We identified and quantitated theY′ bile acid binder, i.e., 3α-hydroxysteroid dehydrogenase (3α-HSD), in rat small intestinal mucosa, and compared its longitudinal distribution with that of glutathione S-transferases (GST). The enzyme activity of 3α-HSD in intestinal mucosa was approximately one-third of that in liver, and it had similar activity in the proximal, middle, and distal portions of the intestine. Immunoreactive protein corresponding to hepatic bile acid binder was detected in all segments of rat small intestine mucosal cytosol by Western blot analysis. There was no significant difference in the concentration of bile acid binder, assayed by enzyme-linked immunosorbent assay (ELISA), between the proximal and the distal intestine, this being 2.93±0.03 and 3.29±0.95 nmol/g tissue, respectively (mean±SD of four animals). On the other hand, the concentration of GST 1-1 showed sharp longitudinal decline and that of GST 3–4 was negligible in the small intestine, as we previously reported, indicating that bile acid binder was a prominent cytosol binding protein in the distal intestine. These results suggested the possible role of bile acid binder in the intracellular transport of bile acids in the ileum.     

Morphological changes of the small-intestinal mucosa of guinea pig and hamster following incubation in vitro and perfusion in vivo with unconjugated bile salts

Incubation in vitro of the intestine of the hamster and guinea pig with 5 mM sodium cholate and with 2 mM sodium deoxycholate and sodium chenodeoxycholate resulted in significant morphological changes compared with control incubations. Generally, no major differences were observed between proximal and distal small intestine or between the species used. Only when guinea pig intestine was incubated with 5 mM cholate was less damage found proximally than distally.Perfusion in vivo of the intestine of the hamster and guinea pig with Krebs-Ringer phosphate results in separation of the epithelium from the lamina propria without excessive shedding of epithelial cells from villous tips. This change was also seen in specimens taken before perfusion and probably represents unavoidable trauma during handling of the intestine.In contrast to studies in vitro, regional differences are readily demonstrable with perfusion of bile salts in vivo. Dihydroxy bile salts produce more marked alterations of both proximal and distal small intestine than the trihydroxy bile salt, sodium cholate. Dihydroxy bile salts result in significantly greater alterations in proximal than in distal mucosa.When 5 mM cholate at pH 6.8 is perfused in the guinea pig, absorption occurs approximately 30 times more rapidly from distal than from proximal segments, while in proximal segments 2 mM chenodeoxycholate is absorbed approximately 15 times more rapidly than 5 mM cholate. A correlation is suggested between the morphological alteration produced in the region of the small intestine by a bile acid and the amount of bile salt passing through the cell. Furthermore, it is proposed that the ileal cells may be damaged to a lesser extent by bile acids normally found in that particular species.     

Biotransformation enzymes in human intestine: critical low levels in the colon?

Biotransformation or drug-metabolising enzymes have an important function in the detoxication of ingested toxic, carcinogenic, or tumour promoting compounds. Enzyme activity and isoenzyme composition of three biotransformation systems: glutathione S-transferase, uridine diphosphate-glucuronosyltransferase, and cytochrome P-450 were studied in normal small and large intestinal mucosa from three kidney donors. The activity of most drug-metabolising enzymes decreases slightly from proximal to distal small intestine, whereas in the mucosa of the large intestine a sharp fall in activity was observed. The isoenzyme composition for each of the three biotransformation systems changed from the small to the large intestine. Class Alpha glutathione S-transferases were not expressed in the colon, in contrast to the small intestine where both Alpha and Pi class isoenzymes are present. In addition, with monoclonal antibodies fewer protein bands for UDP-glucuronosyltransferases and cytochrome P-450 were detected in the colon. In the small intestine both isoforms P-450(4) and P-450(5) were present, whereas in the colon only reduced amounts of cytochrome P-450(4) could be visualised. For UDP-glucuronosyltransferase, 53 and 54 kDa proteins could be detected in the small intestine, but in the colon there was only weak staining of the 54 kDa band. In the normal human colon enzymes are less active and there are fewer isoenzymes present in the mucosa than in the small intestine. This implies a lower level of the detoxifying potential in the colon, which might be important in regard to the high rates of carcinogenesis in the colon.     

Bile salt metabolism in tropical sprue

Mean and peak jejunal bile salt concentrations during digestion of a standard fat meal were found to be significantly lower in six Puerto Rican patients with untreated tropical sprue, all of whom had steatorrhoea, than in six asymptomatic subjects who had normal fat absorption. Bile salt pool size and turnover time did not differ significantly in the two groups.It is suggested that bile salt concentrations may be reduced in the proximal small intestine of patients with tropical sprue as a result of excessive dilution by intestinal fluid. The finding of low bile salt concentrations in two asymptomatic subjects indicates that bile salt lack alone may not be sufficient to produce steatorrhoea.     

Eosinophilic gastroenteritis involving the ileocecal area

Summary Eosinophilic gastroenteritis, an idiopathic inflammation of the alimentary canal, is characterized by infiltration of the intestinal wall by eosinophils, massive submucosal edema, and peripheral eosinophilia. It is generally confined to the gastric antrum and proximal small intestine. A young woman had an eosinophilic infiltrate that involved the distal ileum and right colon only. Barium studies showed severe narrowing and shortening of the cecum and ascending colon. Symptoms of intestinal obstruction did not respond satisfactorily to conservative measures. Adhesions over the ileocecal area as well as thickening and induration of the terminal ileum and proximal right colon were found on hemicolectomy. The remaining intestine and the peritoneal cavity were felt to be normal. Histologic examination showed a cellular infiltrate with prominent eosinophils in the mucosa, submucosal edema and fibrosis. During a 40-month follow-up period after the hemicolectomy, the patient has not shown clear evidence of recurrence or extension of the disease to the stomach or proximal small intestine. It is concluded that idiopathic eosinophilic gastroenteritis may primarily involve the ileocecal area. In that location it must be specifically differentiated from intestinal tuberculosis, amebiasis, and Crohn's disease.     

VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation.

BACKGROUND: Alkaline sphingomyelinase, an enzyme found exclusively in bile and the intestinal brush border, hydrolyzes sphingomyelin into ceramide, sphingosine and sphingosine-1-phosphate, thereby inducing epithelial apoptosis. Reduced levels of alkaline sphingomyelinase have been found in premalignant and malignant intestinal epithelia and in ulcerative colitis tissue. Probiotic bacteria can be a source of sphingomyelinase. OBJECTIVE: To determine the effect of VSL#3 probiotic therapy on mucosal levels of alkaline sphingomyelinase, both in a mouse model of colitis and in patients with ulcerative colitis. METHODS: Interleukin-10 gene-deficient (IL10KO) and wild type control mice were treated with VSL#3 (10(9) colony-forming units per day) for three weeks, after which alkaline sphingomyelinase activity was measured in ileal and colonic tissue. As well, 15 patients with ulcerative colitis were treated with VSL#3 (900 billion bacteria two times per day for five weeks). Alkaline sphingomyelinase activity was measured through biopsies and comparison of ulcerative colitis disease activity index scores obtained before and after treatment. RESULTS: Lowered alkaline sphingomyelinase levels were seen in the colon (P=0.02) and ileum (P=0.04) of IL10KO mice, as compared with controls. Treatment of these mice with VSL#3 resulted in upregulation of mucosal alkaline sphingomyelinase activity in both the colon (P=0.04) and the ileum (P=0.01). VSL#3 treatment of human patients who had ulcerative colitis decreased mean (+/- SEM) ulcerative colitis disease activity index scores from 5.3+/-1.8946 to 0.70+/-0.34 (P=0.02) and increased mucosal alkaline sphingomyelinase activity. CONCLUSION: Mucosal alkaline sphingomyelinase activity is reduced in the intestine of IL10KO mice with colitis and in humans with ulcerative colitis. VSL#3 probiotic therapy upregulates mucosal alkaline sphingomyelinase activity.     

Effects of Ursodeoxycholate and Other Bile Salts on Levels of Rat Intestinal Alkaline Sphingomyelinase (A Potential Implication in Tumorigenesis)

Previous studies showed that bile salts had apromoting effect on colon cancer development and thiseffect was inhibited by ursodeoxycholate (UDC). Werecently found that both human colorectal adenomas and carcinomas were associated with a specificdecrease in alkaline sphingomyelinase activity. In thiswork, we compared the effects of ursodeoxycholate andother bile salts on the levels of rat intestinal alkaline sphingomyelinase both in theintestinal loops and after oral administration. Bilesalts at different concentrations were injected intointestinal loops and the dissociation of alkalinesphingomyelinase from the mucosa was assayed. We found that bilesalts, including taurocholate, taurodeoxycholate,glycocholate, glycochenodeoxycholate, and3-(3-cholamidopropyl dimethylammonio)-1-propanesulonate(CHAPS), dose dependently dissociated alkalinesphingomyelinase from the intestinal mucosa. UDC alonedid not dissociate the enzyme but significantlyinhibited the dissociation caused by other bile saltsand CHAPS. Feeding rats with 0.3% (w/w) taurocholate forfour days decreased peak activity of intestinal alkalinesphingomyelinase by 39% and total activity in theintestine by 20% and increased the output of the enzyme in the feces. In contrast, feeding 0.3%(w/w) UDC for four days increased the peak activity ofalkaline sphingomyelinase in the small intestine by 87%and the activity in the colon by 187% . The total activity of alkaline sphingomyelinase wasincreased by 80% and the output of the enzyme in thefeceswas only slightly increased by UDC administration.The changes in alkaline phosphatase after feeding taurocholate and UDC were much smaller. Ourresults indicate that UDC and other bile salts havedifferent effects on the levels of alkalinesphingomyelinase, which may be implicated in theirdifferent influences on cancer development reportedpreviously.     

Risk factors for small intestinal adenocarcinomas that are common in the proximal small intestine

The frequency of primary small intestinal adenocarcinoma is increasing but is still low. Its frequency is approximately 3% of that of colorectal adenocarcinoma. Considering that the small intestine occupies 90% of the surface area of the gastrointestinal tract, small intestinal adenocarcinoma is very rare. The main site of small intestinal adenocarcinoma is the proximal small intestine. Based on this characteristic, dietary animal proteins/lipids and bile concentrations are implicated and reported to be involved in carcinogenesis. Since most nutrients are absorbed in the proximal small intestine, the effect of absorbable intestinal content is a suitable explanation for why small intestinal adenocarcinoma is more common in the proximal small intestine. The proportion of aerobic bacteria is high in the proximal small intestine, but the absolute number of bacteria is low. In addition, the length and density of villi are greater in the proximal small intestine. However, the involvement of villi is considered to be low because the number of small intestinal adenocarcinomas is much smaller than that of colorectal adenocarcinomas. On the other hand, the reason for the low incidence of small intestinal adenocarcinoma in the distal small intestine may be that immune organs reside there. Genetic and disease factors increase the likelihood of small intestinal adenocarcinoma. In carcinogenesis experiments in which the positions of the small and large intestines were exchanged, tumors still occurred in the large intestinal mucosa more often. In other words, the influence of the intestinal contents is small, and there is a large difference in epithelial properties between the small intestine and the large intestine. In conclusion, small intestinal adenocarcinoma is rare compared to large intestinal adenocarcinoma due to the nature of the epithelium. It is reasonable to assume that diet is a trigger for small intestinal adenocarcinoma.     

Folic acid transport in organ-cultured mucosa of human intestine. Evidence for distinct carriers

The transport of folic acid was investigated in organ-cultured endoscopic biopsy specimens of intestinal mucosa from normal subjects. In the proximal small intestine at pH 5.5 and 6.5, [3H]folic acid accumulated to concentrations 2.64- and 2.17-fold higher than those of the medium, respectively, but at pH 7.5 the concentration was same as that of the medium. Saturability of initial rates of uptake was demonstrable with respect to luminal concentrations of [H+] and folic acid. Two proton-dependent transport mechanisms were evident: (a) a carrier with a low affinity for H+ and a low capacity for folic acid, and (b) a high-affinity, high-capacity carrier (Km, 635 and 54.6 nmol/L [H+]; Vmax, 0.066 and 2.583 pmol folic acid/microL intracellular water per 10 minutes, respectively). Kinetic studies of folic acid uptake at pH 5.5 and 7.5 revealed a difference in Km for folic acid (15.76 and 34.38 mumol/L, respectively) with no change in Vmax. In the colon, folic acid did not accumulate against a concentration gradient, and the initial rate of uptake was not affected by luminal pH. The accumulation of folic acid in colonic mucosa was significantly higher at pH 5.5 than at 7.5 and showed significant regional variation, with optimal uptake in the sigmoid colon. Methotrexate inhibited folic acid uptake competitively both in the proximal small intestine and in the cecum (inhibition constant 6.9 and 54 mumol/L, respectively, at pH 5.5). These data indicate the presence of a proton-dependent, active transport of folic acid in the proximal small intestine at pH 5.0-6.5. At pH 7.5 and in the mucosa of the colon, uptake of folic acid proceeds by facilitated diffusion through a low-affinity carrier.     

Purified intestinal alkaline sphingomyelinase inhibits proliferation without inducing apoptosis in HT-29 colon carcinoma cells

Purpose Sphingomyelin (SM) hydrolysis by sphingomyelinase (SMase) has become an important signalling pathway, with the product ceramide implicated in regulation of cell growth, differentiation and apoptosis. Alkaline SMase is specifically located in the intestinal tract. Marked reductions of the enzyme activity have been found in sporadic colorectal carcinomas and in both adenomas and flat mucosa of patients with familial adenomatous polyposis, indicating an anti-proliferative role in colonic cell growth.Methods We examined the effects of a purified alkaline SMase from rat intestine and a bacterial neutral SMase on cell growth parameters in HT-29 colonic carcinoma cells.Results Alkaline SMase was found to inhibit proliferation of HT-29 cells in both dose-dependent and time-dependent manners. The threshold concentration of the enzyme was approximately 2.5 U/ml, and the maximum effect was obtained at approximately 20 U/ml, which inhibited the cell growth by 50%. The inhibition occurred rapidly, and maximum effect was reached after 12 h of incubation. Dose-dependent inhibition of DNA synthesis was also demonstrated. The effect of alkaline SMase was preceded and accompanied by increased hydrolysis of SM and production of ceramide. Neutral SMase with equivalent hydrolytic capacity did not inhibit cell growth. Alkaline SMase did not induce apoptosis in HT-29 cells. Alkaline SMase did not inhibit growth of IEC-6 cells.Conclusion Alkaline SMase, at doses that induce SM hydrolysis, inhibits growth of colon cancer cells. The inhibition is attributed to an anti-proliferative effect rather than an apoptotic effect.     

Immunohistochemical localisation of vitamin B12 R-binder in the human digestive tract.

The distribution of vitamin B12 R-binder in the human digestive tract was studied using an indirect immunoperoxidase technique. Positive staining for R-binder was found in the mucous cells and ductal epithelial cells of the salivary glands and the oesophageal glands. In normal gastric mucosa, no positive staining for R-binder was found, but in the area with intestinal metaplasia, the columnar epithelial cells and goblet cells showed positive staining. Epithelial cells of the gallbladder, intrahepatic bile ducts and pancreatic ducts were also positive for R-binder. In the small intestine and colon, R-binder was found in the columnar epithelial cells and goblet cells. The measurement of unsaturated vitamin B12 binding capacity and cobalamin content in the extracts from intestinal mucosa also indicated the presence of R-binder in the intestinal mucosa.     

Reduction in alkaline sphingomyelinase in colorectal tumorigenesis is not related to the APC gene mutation

BACKGROUND AND AIMS. The sphingomyelin pathway is an important intracellular mechanism in regulating cell growth. The first step in this pathway is catalysed by sphingomyelinases. Alkaline sphingomyelinase is specifically located in the intestinal tract. Markedly reduced alkaline sphingomyelinase activities have been found in sporadic colorectal tumours and in familial adenomatous polyposis adenomas. Since the adenomatous polyposis coli (APC) gene is mutated in about 80% of sporadic colorectal tumors, and familial adenomatous polyposis is the consequence of a germline mutation of the same gene, we examined whether low alkaline sphingomyelinase activity is linked to APC gene mutations. PATIENTS AND METHODS. Both germline and sporadic adenomatous polyposis coli gene mutations were studied. Alkaline, neutral, and acid sphingomyelinase activities were measured in the intestinal mucosa and content of multiple intestinal neoplasia mice, a murine model of familial adenomatous polyposis and compared to control mice. Alkaline sphingomyelinase activity was also measured in 11 human rectal tumors with APC gene mutation and compared with 9 control tumors without mutation. RESULTS. Alkaline, neutral, and acid sphingomyelinase activities were present in the small intestine and colon in both mice types with no differences in hydrolytic capacity or distribution pattern. In sporadic rectal tumors similar alkaline sphingomyelinase activities were identified in tumors with somatic APC gene mutations as in samples without mutations. In the tumors without detectable APC mutations beta-catenin was analyzed, but no mutation was detected. CONCLUSION. Alkaline sphingomyelinase is not directly linked to adenomatous polyposis coli gene mutations.     

Exogenous sphingomyelinase causes impaired intestinal epithelial barrier function

AIM: To test the hypothesis that hydrolysis of sphingomyelin to ceramide changes the composition of tight junctions (TJs) with increasing permeability of the intestinal epithelium. METHODS: Monolayers of Caco-2 cells were used as an in vitro model for the intestinal barrier. Permeability was determined by quantifi cation of transepithelialflux and transepithelial resistance. Sphingolipid-rich membrane microdomains were isolated by a discontinuous sucrose gradient and characterized by Western-blot. Lipid content of microdomains was analysed by tandem mass spectrometry. Ceramide was subcellularly localized by immunofluorescent staining.RESULTS: Exogenous sphingomyelinase increased transepithelial permeability and decreased transepithelial resistance at concentrations as low as 0.01 U/mL. Lipid analysis showed rapid accumulation of ceramide in the membrane fractions containing occludin and claudin-4, representing TJs. In these fractions we observed a concomitant decrease of sphingomyelin and cholesterol with increasing concentrations of ceramide. Immunofluorescent staining confirmed clustering of ceramide at the sites of cell-cell contacts. Neutralization of surface ceramide prevented the permeability-increase induced by platelet activating factor. CONCLUSION: Our findings indicate that changes in lipid composition of TJs impair epithelial barrier functions. Generation of ceramide by sphingomyelinases might contribute to disturbed barrier function seen in diseases such as inflammatory, infectious, toxic or radiogenic bowel disease.     

Regulation of intestinal concentration of cholecystokinin by bile and/or pancreatic juice

Pancreatic exocrine secretion in conscious rats is regulated by intraluminal bile and/or pancreatic juice. Exclusion of bile and/or pancreatic juice from the intestinal lumen caused cholecystokinin (CCK) release and stimulated pancreatic secretion. CCK in the plasma is mainly derived from endocrine cells in the proximal small intestinal mucosa. We examined the changes in CCK concentrations in the intestinal mucosa and compared them to those of plasma CCK concentrations and the changes of luminal trypsin activities after bile and/or pancreatic juice diversion in conscious rats. Rats with bile and pancreatic fistulae were used. Each treatment of bile, pancreatic juice, and bile-pancreatic juice diversion decreased luminal trypsin activity and increased plasma and intestinal CCK concentrations. The potency of the stimulatory effect on plasma and intestinal CCK concentrations was bilepancreatic juice diversion>pancreatic juice diversionbile diversion. Neither plasma CCK concentration nor intestinal CCK concentration was in inverse proportion to trypsin activity. The plasma CCK concentration did not parallel intestinal CCK concentration. Intravenous infusion of CCK-8 (300 pmol/kg/hr) did not increase CCK concentration in the intestinal mucosa. It was proposed that bile and/or pancreatic juice in the intestinal lumen regulated CCK concentrations not only in the plasma but also in the intestinal mucosa.     

Endocrine regulation of ion transport in the avian lower intestine

The lower intestine (colon and coprodeum) of the domestic fowl maintains a very active, transporting epithelium, with a microvillus brush border, columnar epithelial cells, and a variety of transport systems. The colon of normal or high salt-acclimated hens expresses sodium-linked glucose and amino acid cotransporters, while the coprodeum is relatively inactive. Following acclimation to low salt diets, however, both colon and coprodeum shift to a pattern of high expression of electrogenic sodium channels, and the colonic cotransporter activity is simultaneously downregulated. These changes in the transport patterns seem to be regulated, at least in part, by aldosterone. Our recent work with this tissue has focused on whether aldosterone alone can account for the low salt pattern of transport. Other work has looked at the changes in morphology and in proportions of cell types that occur during chronic acclimation to high or low salt diets, and on a cAMP-activated chloride secretion pathway. Recent findings suggesting effects of other hormones on lower intestinal transport are also presented.     

Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease

AIM: To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease. METHODS: Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn's disease(n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps(n = 6) and colorectal cancer(n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1 a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine(PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A(HEXA) and sialidase-3(NEU3) were measured in intestinal mucosa using western blot and compared among subject groups. RESULTS: Relative GM3 ganglioside content was 2-fold higher(P 0.05) in intestine from patients with inflammatory bowel disease(IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue(P 0.05). Control intestine exhibited 3-fold higher(P 0.01) relative GD1 a ganglioside content than IBD intestine. GD3 and GD1 a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine(P 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold(P 0.05) and NEU3 was increased 8.3-fold(P 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1 a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1 a and PC.CONCLUSION: This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.     

Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha.

Recent investigations have identified a signal-transduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinase generates ceramide, which may be converted to the protein kinase C inhibitor sphingosine or to ceramide 1-phosphate. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-431 cells. The present studies describe a kinase that may mediate ceramide action. With a 19-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphorylated the peptide was detected in A-431 cells. Activity was linearly related to ATP (0.3-300 microM) and peptide concentration (0.02-1 mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg(2+)-dependent. Other cations--Ca2+, Mn2+, and Zn(2+)--were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 microM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least 15 min. As little as 0.001 microM ceramide was effective, and 1 microM ceramide induced maximal phosphorylation. Sphingosine was similarly effective. Because tumor necrosis factor (TNF) alpha rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased 1.5-fold at 5 min and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg(2+)-dependent ceramide-activated protein kinase that may mediate some aspects of TNF-alpha function.     

Intestinal disaccharidase activity following pancreatic duct occlusion in the rat

The influence of pancreatic secretions on growth and brush-border enzyme activity, throughout the entire small intestine, was examined in the rat. Pancreatic secretions were excluded from the gut lumen by stapling the pancreatic ducts, without interruption of bile flow. The entire small intestine was studied as four segments; the duodenum and three distal segments of equal length. Weight of intestine and mucosa, and mucosal sucrase, isomaltase, lactase, and alkaline phosphatase activity were measured 10-15 days following pancreatic duct occlusion, or sham-operation. The duodenum of pancreatic duct-occluded animals exhibited significant hypertrophy. In general, specific and total disaccharidase activities were greater in duct-occluded animals than in controls throughout the intestine. The increase was more pronounced in distal than in proximal segments. The sucrase/isomaltase ratio was significantly greater in pancreatic duct-occluded animals than in controls in the two distal segments. Alkaline phosphatase activity was not affected by pancreatic duct occlusion. The greater relative increase of disaccharidase activities and sucrase/isomaltase activity ratios in the distal segments of duct-occluded animals, indicates a more important regulatory role of pancreatic enzymes in the distal small intestine. It is concluded that regulation of intestinal brush-border enzyme activity by pancreatic secretion is selective for enzyme and site as follows: disaccharidases, but not alkaline phosphatase, are regulated; the sucrase subunit of the sucrase/isomaltase complex is most sensitive to regulation, while lactase is least sensitive; and the regulatory effect on disaccharidases is greater in distal than in proximal intestine.     

Fat digestion and exocrine pancreatic function in primary biliary cirrhosis

Intestinal bile salt deficiency marginally impairs fat absorption in cholestatic patients. The finding of massive steatorrhea in some patients with primary biliary cirrhosis led us to systematically study and compare fat digestion in control subjects (n = 4) and patients with biliary cirrhosis with (n = 11) and without (n = 3) steatorrhea. The jejunum was anatomically and functionally intact in all subjects, as demonstrated by normal gastrointestinal radiology and xylose absorption, respectively. The intestinal contents were recovered during digestion of a fat meal. Lipolysis, pH, and trypsin activity were measured in whole intestinal contents, whereas bile salts, total lipid, and fatty acid were determined in both total and aqueous phases. The results obtained in controls and patients without steatorrhea were similar. Percentage of lipolysis and intraluminal pH were normal in controls and in both patient groups. The intestinal contents of the patients with steatorrhea had a significantly lesser capacity to solubilize both total lipid and fatty acid in relation to abnormally low aqueous bile salt concentrations. No bile salt deconjugation and only minimal bile salt precipitation were found, thus low aqueous bile salts were strictly related to bile secretory failure. Steatorrhea was always present when aqueous bile salt levels were below 3.0 mM. Intestinal trypsin activity was subnormal in patients with steatorrhea; decreased trypsin activity was related (r = 0.82, p less than 0.001) to reduced intestinal bile salt levels. One patient was found to have severe exocrine pancreatic failure. Administration of medium chain triglycerides was uniformly effective in improving nutrition in patients with steatorrhea, but the course of the disease was unaffected. These results indicate that overt pancreatic failure is uncommon in primary biliary cirrhosis, and that fat maldigestion and steatorrhea, regardless of what degree, are due mainly to low intestinal bile salt levels secondary to bile secretory failure. Finally, subnormal pancreatic function in this disease appears to be related to the bile secretory failure, suggesting either that the lack of bile or bile salts in the intestine depresses pancreatic exocrine function or that both biliary and pancreatic secretions decrease in parallel as part of a widespread secretory failure syndrome.