Biomedical subjects

K F Sewing

Publications and source records attributed to K F Sewing.

At least 37 records · Page 2Linked to original sources

PubMed1998

Structural elucidation by electrospray mass spectrometry: an approach to the in vitro metabolism of the macrolide immunosuppressant SDZ RAD.

SDZ RAD [40-O-(2-hydroxyethyl)rapamycin] is a macrolide immunosuppressant that is currently under clinical investigation after organ transplantation. The elucidation of its metabolic pathway is essential to improve the understanding of its therapeutic potentials and safety. In this article we describe investigations on the structural identification of some major metabolites of the drug produced by human liver microsomes in vitro. The principles described may be generally applicable for the structural elucidation of complex compound mixtures in biological matrices. Under the conditions of electron impact ionization, SDZ RAD undergoes extensive fragmentation and no information sufficient for structural elucidation is obtained. Therefore, mass spectrometry based on soft electrospray ionization (ESI) in conjunction with collision-induced fragmentation was the method of choice. High-performance liquid chromatography coupled to an ESI mass spectrometer resulted in separation and identification of 16-O-demethyl-SDZ RAD, the ring-opened form of SDZ RAD, and its dehydrate. Additionally, we characterized several demethylated and hydroxylated metabolites.

Chromatography, High Pressure Liquid↗

PubMed1998

The Helicobacter pylori fatty acid cis-9,10-methyleneoctadecanoic acid stimulates protein kinase C and increases DNA synthesis of gastric HM02 cells.

Protein kinase C (PKC) has been implicated in the control of epithelial proliferative activity and in the process of malignant transformation. Helicobacter pylori (H.p.) infection is associated with increased gastric epithelial cell proliferation and has been linked with gastric carcinoma. In the present study, we report that the H.p. fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) directly activates PKC (Ka 3.3 microM). The effect of MOA upon PKC activation was Ca2+ dependent but did not require phosphatidylserine as phospholipid cofactor. MOA increased the stimulatory effect of phosphatidylserine at low Ca2+ (1 microM) concentrations. These findings indicate that MOA interacts at the phospholipid- and the diacylglycerol-binding domain to elicit PKC activation. Treatment of gastric mucous cells HM02 caused translocation of PKC from the cytosol to the nuclear, mitochondrial and membrane fraction. Furthermore, MOA stimulated [3H]thymidine incorporation into the DNA of HM02 cells. Our results show that the H.p. fatty acid MOA activates PKC and increases DNA synthesis in gastric epithelial cells.

Animals↗

PubMed1998

Metabolism and transport of the macrolide immunosuppressant sirolimus in the small intestine.

Small intestinal metabolism and transport of sirolimus, a macrolide immunosuppressant with a low and highly variable oral bioavailability, were investigated using small intestinal microsomes and intestinal mucosa in the Ussing chamber. After incubation of sirolimus with human and pig small intestinal microsomes, five metabolites were detected using high performance liquid chromatography/electrospray-mass spectrometry: hydroxy, dihydroxy, trihydroxy, desmethyl and didesmethyl sirolimus. The same metabolites were generated by human liver microsomes and pig small intestinal mucosa in the Ussing chamber. Anti-CYP3A antibodies, as well as the specific CYP3A inhibitors troleandomycin and erythromycin, inhibited small intestinal metabolism of sirolimus, confirming that, as in the liver, CYP3A enzymes are responsible for sirolimus metabolism in the small intestine. Of 32 drugs tested, only known CYP3A substrates inhibited sirolimus intestinal metabolism with inhibitor constants (Ki) equal to those in human liver microsomes. The formation of hydroxy sirolimus by small intestinal microsomes isolated from 14 different patients ranged from 28 to 220 pmol.min-1.mg-1 microsomal protein. In the Ussing chamber, >99% of the sirolimus metabolites reentered the mucosa chamber against a sirolimus gradient, indicating active countertransport. Intestinal drug metabolism and countertransport into the gut lumen, drug interactions with CYP3A substrates and inhibitors in the small intestine and an 8-fold interindividual variability of the intestinal metabolite formation rate significantly contribute to the low and highly variable bioavailability of sirolimus.

Animals↗

PubMed1998

Automated simultaneous quantification of the immunosuppressants 40-O-(2-hydroxyethyl) rapamycin and cyclosporine in blood with electrospray-mass spectrometric detection.

A new analytical method to quantify 40-O-(2-hydroxyethyl)rapamycin (SDZ RAD) and cyclosporine (Cs) simultaneously in blood is presented. The combination of an on-line solid-phase extraction step with an HPLC system coupled to an electrospray mass spectrometer gave excellent specificity, sensitivity, and reproducibility. Aliquots of deproteinized blood samples were injected into the HPLC system and extracted on-line, using a conventional C18 guard column. The extract was eluted from the guard column in the backflush mode and injected into the liquid chromatography-mass spectrometry system. The calibration functions for SDZ RAD and Cs extracted from blood with added analyte were linear from 0.15 to 30 microg/L (r2 = 0.999) and from 1.5 to 1000 microg/L (r2 = 0.999), respectively. The CVs of peak areas were 6.2% at 10 microg/L SDZ RAD (n = 6) and 6.2% at 100 microg/L Cs (n = 6). Recovery ranged from 84.3% to 102.3% for SDZ RAD and from 81.7% to 92.2% for Cs. The lower limit of detection for both drugs was 0.05 microg/L. A rate of four samples per hour was maintained during the consecutive analysis of SDZ RAD and Cs in >500 blood samples with one single extraction and analytical column. The method described is a powerful tool for the simultaneous determination of SDZ RAD and Cs in blood. It works without time-consuming sample preparation steps and with excellent reproducibility. Because of the detection performance of electrospray mass spectrometry, this system offers flexibility in the working range, which is essential for therapeutic drug monitoring under different conditions.

Chromatography, High Pressure Liquid↗

PubMed1997

Pepsinogen synthesis during long-term culture of porcine chief cells.

The purpose of this study was to characterize time-dependent changes in pepsinogen (PG) synthesis of porcine gastric chief cells during long-term monolayer culture. Porcine chief cells were isolated by pronase/collagenase treatment of fundic mucosa and enriched by density gradient and counterflow centrifugation. PG isoenzymes were identified in [L-35S]methionine-labelled cultured chief cells by native polyacrylamide gel electrophoresis followed by phosphor imager analysis, protease detection and immunoblots with specific PG A and C antibodies. The obtained results suggest that porcine chief cell cultures, after an initial settling period, reached an approximate steady state in total protein content and synthesis as well as in PG content and isoenzyme pattern from days 3 to 9 of culture. The latter was characterized by the presence of at least two PG A and two PG C isoenzymes. During the supposed steady-state total PG synthesis averaged out at 34 +/- 2% of total protein synthesis, as detected by [L-35S]methionine incorporation, due to the synthesis of, mainly, PG A2 and, to a much lesser extent, PG C and A1. In line with an active secretion, PG A2 proportion was on average significantly higher in released (44 +/- 3%) than in intracellular labelled proteins (19 +/- 2%). In addition, PG release from chief cells cultured for 6 and 9 days could be stimulated by cholecystokinin-octapeptide. These data suggest that porcine chief cells in monolayer culture are a model well suited for the quantitative and qualitative characterization of PG isoenzyme synthesis and release during long-term investigations, for which an establishment of a culture steady state appears to be a useful prerequisite.

Animals↗

PubMed1997

Drug metabolism in hepatocyte sandwich cultures of rats and humans.

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.

Adult↗

PubMed1997

Pitfalls in monitoring tacrolimus (FK 506).

Tacrolimus (FK 506) is a new, potent immunosuppressive drug for primary and rescue therapy in liver and kidney transplantation. Therapeutic drug monitoring is essential for this drug because of its narrow therapeutic window. Blood levels are monitored routinely by enzyme linked immunoassay (ELISA) or by microparticle enzyme immunoassay (MEIA). In a 13-year-old recipient of a liver transplant who had poor hepatic function during the first postoperative week, the authors observed unusually high tacrolimus blood concentrations using either the ELISA (26.6 to 49.0 microg/l) or MEIA (58.5 to 64.5 microg/l). Parent drug levels measured in the same blood samples by high-performance liquid chromatography/mass spectrometry (HPLC/MS) were up to 10-fold lower (5.1 to 9.0 microg/l). The discrepancies between the immunoassay and HPLC/MS results could not be attributed to any of the known metabolites of tacrolimus.

Adolescent↗

PubMed1997

Recovery of cholecystokinin response of porcine gastric chief cells during monolayer culture.

Cell isolation may impair secretory chief cell functions. To evaluate whether a monolayer culture results in a recovery, we compared the effects of cholecystokinin (CCK) octapeptide (CCK-8) on pepsinogen release from freshly isolated and from cultured porcine chief cells. CCK-8 had no significant effect on freshly isolated porcine chief cells but stimulated pepsinogen release from 36- and 60-hour cultured cells with EC50 values of 180 and 130 nmol/l, respectively. Maximal stimulation, achieved at a concentration of 1 micromol/l, amounted to 289 +/- 63 (p <0.01) and 401 +/- 64% (p <0.01) of the respective control value. In addition, the CCK-8 concentration-response curve for 60-hour, but not for 36-hour cultured chief cells displayed a second stimulatory peak at a CCK-8 concentration of 100 pmol/l (266 +/- 55% of control value, p < 0.05) with an EC50 value of 16 pmol/l. The CCKA-receptor antagonist devazepide (10 nmol/l) prevented the stimulatory effect of 1 micromol/l CCK-8 on pepsinogen release of 60-hour cultured cells. The adenylate cyclase activator forskolin (10 micromol/l) potentiated the low concentration CCK-8 effect, shifting the peak stimulation to a CCK-8 concentration of 10 pmol/l, and inhibited the high concentration CCK-8 effect on 60-hour cultured cells. These results indicate a time-dependent recovery of the CCK response of porcine gastric chief cells in monolayer culture and suggest that this model has an advantage over freshly isolated chief cells with regard to the pharmacological characterization of CCK effects.

Adenylyl Cyclases↗

PubMed1996

3-D coculture of hepatic sinusoidal cells with primary hepatocytes-design of an organotypical model.

Models for cocultures of parenchymal (PC) and nonparenchymal cells (NPC) of the liver relied on mixing the cells in a two-dimensional configuration or on establishing spheroidal aggregates. In vivo hepatic nonparenchymal cells, such as endothelial cells and Kupffer cells, are separated from parenchymal cells by extracellular matrix (ECM). Due to their location outside of the space of Disse they can form a barrier toward the sinusoid. Hepatocytes are attached to ECM of the space of Disse via two opposing sinusoidal surfaces. No three-dimensional coculture model reflecting this specific microenvironment of the liver cell plates in vivo has been available to date. We designed a three-dimensional model by positioning NPC on top of PC enclosed as a monolayer within a collagen sandwich. A gas-permeable membrane support can be used to allow the supply of oxygen to the resulting cell plate also from underneath the cell layers. Morphological analysis was performed by inverse and cross-sectional studies by light microscopy, scanning, and transmission electron microscopy of the coculture model. Cuboidal hepatocytes formed confluent layers below the NPC layer. They regularly expressed bile canaliculi at intercellular contact zones. Both sinusoidal surfaces expressed microprojections. Characteristic NPC including endothelial cells, Kupffer cells, and Ito cells completely covered the second matrix layer within a week. Kupffer cells were located on top of endothelial cells. Ito cells were intermingled and could be identified by their intracytoplasmic lipid droplets. LPS stimulation of cocultures resulted in a depression of albumin secretion. Phase I and phase II metabolites of the cytochrome P-450 1A1 substrate ethoxyresorufin were generated independently from the presence of cocultured NPC. This study describes the development of a novel three-dimensional coculture model, which intends to mimic more closely the microenvironment of the hepatic sinusoid by respecting the specific plate structure of the liver parenchyma. The model could serve as a complex tool to study potential collaborations between PC and NPC of the liver.

Albumins↗

PubMed1996

Tacrolimus (FK 506) biotransformation in primary rat hepatocytes depends on extracellular matrix geometry.

Established in vitro models for studies of hepatic drug biotransformation include the use of primary hepatocytes. In normal liver the space of Disse provides the possibility of bilateral attachment to extracellular matrix for each hepatocyte. This configuration is disrupted by the cell isolation procedure of normal liver tissue, which delivers suspensions of round shaped cells. In standard culture configurations this unphysiologic cell shape terminates in a morphological dedifferentiation and inability to biotransform drugs. This study analyses the relevance of extracellular matrix geometry in hepatocyte monolayer configurations for expression and activity of cytochrome P450 3A. This enzyme is involved in the biotransformation of a large number of pharmaceuticals including the immunosuppressants tacrolimus and sirolimus. Morphological analysis of primary rat hepatocytes cultured with and without overlay of collagen type I was performed by transmission and scanning electron microscopy. Expression and activity of cytochrome P450 3A was studied by Western blot and the use of two model drugs specific for this enzyme. To this purpose the immunosuppressive drugs tacrolimus and sirolimus were used. Metabolites were analyzed by HPLC and HPLC/MS. Two sided attachment to extracellular matrix induces profound changes of the hepatocellular morphology in vitro resulting in the reconstitution of a polyhedric cell shape. This phenomenon is paralleled by an enhanced expression of cytochrome P450 3A and corresponding metabolic activity. As shown for tacrolimus biotransformation, the model may be useful to study complex metabolic patterns. In addition this model may facilitate studies of the kinetics of hepatocellular drug biotransformation in a setting with prolonged stability.

Albumins↗

PubMed1996

Rat gastric hydrophobic barrier: modulation of phosphatidylcholine molecular species by dietary lipids.

Phospholipids protect the gastric mucosa by forming a proton-repellent hydrophobic layer on its luminal surface. We have recently shown that two molecular species of phosphatidylcholine (PC), PC16:0/18:1, and PC16:0/18:2, but not PC16:0/16:0, are predominantly released into gastric mucus. We investigated whether these molecular species in mucus are modified by dietary fat. Rats were fed (for three weeks) a diet supplemented with either 10% cod liver, palm, or sunflower oil, or 10% corn starch as a control. In tissue, cod liver oil decreased PC16:0/20:4 and PC18:0/20:4. Cod liver oil and palm oil increased PC16:0/18:1, whereas sunflower oil decreased PC16:0/18:1. Palm oil additionally decreased PC16:0/18:2, whereas the other diets had no effect on PC16:0/18:2. In mucus, however, PC16:0/18:1 and PC16:0/18:2 were not significantly altered by any diet. They were increased over tissue values and comprised 37.6 +/- 3.3 and 33.1 +/- 1.4 mol% in controls. PC16:0/16:0 was lower in mucus than in mucosa and even decreased by cod liver oil (1.2 +/- 0.2 vs. 2.7 +/- 0.3 mol%; P < 0.01). We conclude that PC16:0/18:1 and PC16:0/18:2 are modified by dietary fat in tissue. In gastric secretions, however, PC16:0/18:1 and PC16:0/18:2 are kept constant and together comprise 70 mol% of the released PC species, whereas PC16:0/16:0 does not play a role for the gastric hydrophobic barrier under any dietary treatment. Additionally, cod liver oil decreases the content of PC16:0/20:4 and PC18:0/20:4 in gastric mucosa, thereby possibly decreasing the formation of eicosanoids.

Animals↗

PubMed1996

Cultured gastric parietal cells from the guinea pig: adherence, cell growth and stimulus coupling of Ca2+ and cyclic AMP.

The aim of the study was to establish cell culture conditions for responsive guinea pig parietal cells. Parietal cells were isolated by a pronase/collagenase method, enriched by counterflow elutriation and cultured on plastic culture dishes in minimum essential medium. Precoating with gelatine or collagen increased adherence; optimum fetal calf serum concentration was 10%. Parietal cells were cultured for up to 120 h. Intracellular calcium levels in cells cultured for 48 h were 150 nmol/l and increased to 320 nmol/l after stimulation with carbachol and to 250 nmol/l after histamine stimulation as determined by video imaging microscopy. Intracellular cyclic AMP levels were increased 9-fold by histamine in cells cultured for 24 h and more than 30-fold in cells cultured for 48 h. The results show that guinea pig parietal cells grow in primary culture and are suitable for studying second messenger coupling.

Animals↗

PubMed1996

Diltiazem increases blood concentrations of cyclized cyclosporine metabolites resulting in different cyclosporine metabolite patterns in stable male and female renal allograft recipients.

1. Six male and six female stable renal allograft recipients under cyclosporine immunosuppression and without concomitant therapy with drugs known either to induce or inhibit CYP3A enzymes were included in the study and received 180 mg day-1 diltiazem for 1 week in a two-period cross-over fashion. Cyclosporine (352 +/- 56 mg day-1) was given in two daily oral doses. The daily doses were not changed during the study. Blood samples were collected for 12 h after receiving cyclosporine alone and after receiving diltiazem in addition for 1 week. Cyclosporine and nine of its metabolites were quantified using h.p.l.c. 2. Co-administration of diltiazem caused a 1.6 fold increase of the AUC (0, 12 h) of cyclosporine and a 1.7 fold increase of the AUC(0, 12 h) of its metabolites. Analysis of the metabolite patterns showed an over-proportional increase of the AUC(0, 12 h) of the cyclized metabolites AM1c (2.6 fold) and AM1c9 (2.2 fold). The AUC(0, 12 h) values of cyclosporine and the hydroxylated metabolites increased less than two fold. 3. Differences of the AUC(0, 12 h) values of cyclosporine with and without diltiazem were significantly higher in female than in male patients (P < 0.02). The differences in the AUC(0, 12 h) values of the metabolites, especially AM1c, tended to be higher in female patients as well. 4. It is concluded that coadministration of diltiazem not only increases the blood concentration of cyclosporine but also those of its metabolites, leads to a shift of the metabolite pattern towards cyclized metabolites, and that the pharmacokinetic changes under diltiazem administration are more prominent in female than in male patients.

Adult↗

PubMed1996

Synthesis and release of phosphatidylcholine by isolated porcine gastric mucous cells in primary culture.

Phosphatidylcholine (PC) is the major phospholipid of the hydrophobic gastric mucosal barrier and is chiefly released from mucous cells into the gastric mucus. Whereas the mucosa contains highly unsaturated PC, gastric mucus predominantly contains palmitoyl-oleoyl-PC and palmitoyl-linoleoyl-PC, indicating a selective release of these PC species into the gastric lumen. In order to understand gastric PC metabolism, we investigated synthesis and release of PC in cultivated porcine gastric mucous cells, using dual labelling with [methyl-3H]-choline and [1-14C]-palmitate, in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), indomethacin and prostaglandin E2 (PGE2). Linear incorporation of [methyl-3H]-choline and [1-14C]-palmitate into PC was achieved for at least 8h. In contrast to type II pneumocytes TPA increased PC synthesis in gastric mucous cells but not its release. Indomethacin did not influence PC synthesis, but it decreased the release of newly synthesized PC. PGE2 antagonized the effect of indomethacin on PC release. We conclude that PC release by isolated porcine gastric mucous cells is regulated in a manner different from type II pneumocytes. PC release is impaired by indomethacin and this impairment is restored by PGE2.

Animals↗

PubMed1996

Identification of drugs inhibiting the in vitro metabolism of tacrolimus by human liver microsomes.

1. Tacrolimus, an immunosuppressive macrolide, is metabolized by enzymes of the cytochrome P450 3A subfamily. In this study, 34 drugs were tested for their interactions with tacrolimus metabolism by human liver microsomes. 2. Fifteen drugs which inhibit the in vitro metabolism of tacrolimus were identified: bromocriptine, corticosterone, dexamethasone, ergotamine, erythromycin, ethinyloestradiol, josamycin, ketoconazole, miconazole, midazolam, nifedipine, omeprazole, tamoxifen, troleandomycin and verapamil.

Bromocriptine↗

PubMed1996

Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber.

1. The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2. In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy-demethyl tacrolimus were generated. Their formation was concentration- and time-dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3. The metabolite formation was highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. 4. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5. Tacrolimus metabolites re-entered the mucosa chamber (> 90%) and passed through the small intestinal preparation into the serosa chamber. 6. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re-enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus.

Animals↗

PubMed1996

Drug interactions and interindividual variability of ciclosporin metabolism in the small intestine.

The undecapeptide ciclosporin is used as immunosuppressant after organ transplantation and for therapy of immune diseases. Low and variable bioavailability of ciclosporin has been attributed to its metabolism in the small intestine. The aim of the present study was to investigate drug interactions and interindividual variability of ciclosporin metabolism in the small intestine. Ciclosporin metabolism was studied in vitro using microsomes isolated from the small intestine of humans and pigs. The metabolites generated were quantified by HPLC and identified by mass spectrometry. Using specific antibodies and inhibitors, we showed that, as in the liver, cytochrome P450 3A (CYP 3A) enzymes are responsible for ciclosporin metabolism in the human small intestine. Of the 28 xenobiotics included in the study, 16 drugs, all well-known CYP 3A inhibitors, inhibited ciclosporin metabolism in the small intestine. In the small intestine of different patients, the rate of metabolism varied by a factor of 10. Ciclosporin was metabolized faster by small intestine microsomes from female (n = 4) than from male (n = 10) patients (p < 0.009).

Adult↗

PubMed1996

Bicarbonate secretion in the guinea pig duodenum: functional characterization of peptide hormone receptors in duodenal enterocytes.

To get information about the peptide hormone receptors involved in duodenal bicarbonate secretion (DBS) and their cellular location, we determined DBS and adenylate cyclase (AC) activity in response to hormones of the vasoactive intestinal polypeptide (VIP)/secretin family of peptides. DBS was determined in an isolated, perfused (24 mmol/1 NaHCO3) loop of the proximal duodenum in urethane- and indometacin-treated guinea pigs. AC stimulation was measured in isolated, homogenized duodenal enterocytes, the histological evaluation of which revealed their villous origin. VIP (10(-9) to 10(-7) mol x kg-1) dose-dependently increased DBS 3.5-fold (p < 0.01); this effect was completely inhibited by the VIP antagonist [D-p-Cl-Phe6,Leu17]VIP (10(-6) mol x kg-1). Glucagon (10(-8) to 10(-6) mol x kg-1) increased DBS 2.1-fold, while secretin (10(-9) to 10(-6) mol x kg-1) had no effect on DBS, but stimulated pancreatic bicarbonate secretion. VIP concentration-dependently increased AC activity 5.6-fold with an EC50 of 1.3 x 10(-9) mol/l. [D-p-Cl-Phe6,Leu17]VIP caused a rightward shift of the VIP concentration-response curve. A Schild plot analysis yielded a slope of 0.85 +/- 0.11, indicating competitive inhibition. While secretin also stimulated AC activity, although 1,000-fold less potent than VIP, glucagon was ineffective. These data indicate that specific VIP receptors, which mediate VIP-stimulated bicarbonate secretion, are present on villous enterocytes. Stimulation of AC by secretin seems to be of pharmacological relevance only and is consistent with the lack of effect of this hormone on DBS. Glucagon likely activates a second transmitter of bicarbonate secretion, or works independently of AC.

Adenylyl Cyclases↗