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was unaffected. Recently, GnSIF bioactivity was also mea- sured in steroid-free testicular extracts. The aim of the pres- ent research was to investigate a possible role for FSH in the self-priming action of GnRH in the adult male rat.

Adult intact or gonadectomized male and female rats were treated with either 10 IU FSH (MetrodinR, highly purified urinary FSH from post-menopausal women; Ares-Serono) or saline (0.9OYa NaCl (w/v)) on three occasions, starting 2 days before the pulsatile administration of GnRH (500 or 2000 pmol/kg body weight). The administration of GnRH (500 pmol only) in female rats took place on the day of pro-oes- trus. The self-priming capacity was demonstrated during four administrations of GnRH 1 h apart in animals anaesthetized with phenobarbital. Blood samples were collected just before and 20 min after each GnRH stimulation and were used for the estimation of LH. The LH response was expressed as the difference of these two values.

As expected, ovariectomized and orchidectomized rats did not show the GnRH-induced self-priming effect. In intact male rats, the self-priming effect of the highest dosage of GnRH was most pronounced. However, the maximal effect was minor and the timing retarded if compared with the self- priming effect in female rats. In contrast to intact female rats, FSH was ineffective to reduce the initial response as well as to delay the timing of the maximal response to both treat- ments with GnRH in intact male rats. Hence the self-priming potency of GnRH was unaffected by FSH.

The present results point to a possible sex difference in the release and/or mechanism of action by which the self-prim- ing effect of GnRH is established. GnSIF-like activity in the male rat may already be secreted at a maximal rate or this secretion may be less sensitive to the action of FSH. This might be caused by the difference in circulating sex steroids. Otherwise, the GnRH-induced self-priming effects in male and female rats might be two totally different phenomena.

P5 Lymphocyte adhesion to brain capillary endothelial cells in vitro. H.E. de Vriesa, A.C.E. Moor”, M.C.M. Blom-Roosma- lena, A.G. de Beef, J. Kuiperb, Th.J.C. van Berkelb, D.D. BreimelB, Leiden/Amsterdam Center for Drug Research, Di- visions of “Pharmacology and bBiopharmaceutics, Center for Bio-Pharmaceutical Sciences, Leiden University, Sylvius Lab- oratories, P.O.Box 9503, 2300 RA Leiden, The Netherlands

The induction of adhesion molecules on brain capillary en- dothelial cells (BCEC) was investigated in primary cultures. The adhesion of lymphocytes to cerebral endothelial cells was determined using an in vitro lymphocyte adhesion assay. Monolayers of BCEC were incubated with lipopolysacchar- ide ( LPS ) , interleukin- 1 (rhIL- 1) , and interleukin-6 (rhIL- 6) to simulate an inflammatory site in the cerebral capillar- ies. Cerebral endothelial cells were characterized and were positive for factor VIII-related antigen, showed specific up- take of acetylated low density lipoprotein ( AcLDL), con- tained high levels of y-glutamyl transpeptidase (y-GTP) and presented tight junctions.

The adhesion of lymphocytes to BCEC increased 3-4-fold, when endothelial cells were stimulated for 4 h using 5 or 10

ng/ml LPS. In addition, adhesion increased dose-depen- dently with IL-l 1.5-fold (25 ng/ml) to 3-4-fold (100 ng/ ml). BCEC stimulated with IL6 showed also a dose-depen- dent increase in the adhesion of lymphocytes, cells treated with 50 ng of IL-6/ml showed a 2-fold increase, while treat- ment with 100 ng/ml resulted in a 3-fold increase of lympho- cytes adherence. Stimulation with 5 ng/ml IL-6 overnight re- sulted in a 3-4-fold increase of attachment. Specific monoclonal antibodies directes against CD1 la, CD18 and VLA4 were used to inhibit the adherence of the lymphocytes in this bioassay. It was determined that intercellular adhe- sion molecule (ICAM- ) and vascular adhesion molecule (VCAM) are involved in the adhesion of lymphocytes to BCEC.

The results show that it now may be possible to simulate an inflammation site in our in vitro model for the blood-brain barrier. It was shown that lymphocyte adhesion to cerebral endothelium could be induced by LPS, IL-l or IL-6 and was mediated by ICAM- and VCAM.

P6 Small intestinal paracellular permeability include macro- molecules with molecular weights around 10000 Da. N. Pan- tzala, B.R. WestrGma and S. Lundinb, “Lund University, De- partment ofAnimal Physiology, Helgonaviigen 3B, S-223 62 Lund, Sweden, bLund University Hospital, Department of Clinical Pharmacology S-221 85 Lund, Sweden

Background: The purpose with this investigation was to characterize the size-limit for macromolecules subjected to paracellular permeation through the small-intestinal epithe- lium, which could have implications on the oral delivery of protein and peptide drugs. We have previously used differ- ent-sized macromolecules in the 3000-70000 Da range in or- der to study permeation routes for macromolecules through the epithelium in vitro in diffusion chambers [ 11. By using this model with different-sized markers, together with a cir- cumvention of the normally existing waterflux through the epithelium by applying an osmotic load on the mucosal side [ 2 1, light could be shed on the question at issue.

Materials and Methods: Small intestinal segments from Sprague Dawley rats were mounted in diffusion chambers [ 31 and 5 ml modified Krebs buffer was tilled on both the mu- cosal and serosal side. The buffer was continuously oxygen- ated, circulated and tempered to 37°C. In this system FITC- and RITC-dextrans with M,,,s of 3000, 10000, 40000 and 70000 Da were introduced on the mucosal side as permeabil- ity markers. The intestinal permeability was determined by measuring the marker appearance on the serosal side during 2 h. This was performed both at normal osmotic pressure, i.e., 330 mOsm/l in the Krebs buffer, and when an osmotic load of 1000 mOsm/l was applied on the mucosal side by the addition of polyethylene glycol600.

Results: The permeability during normal osmotic pressure was dependent on the molecular size of the marker; the smallest molecule dextran 3000 showed the highest permea- tion, while the largest, dextran 70000, showed the lowest per- meation. The result of the changed waterflux through the ep- ithelium by applying an osmotic pressure of 1000 mOsm/l

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caused a decreased permeability to dextrans with M,s of 3000 and 10000 Da, however, still being higher than the permea- bility to the larger dextrans, while the permeability to 40000 and 70000 Da dextrans was unchanged or increased.

Conclusions: The dextrans with Mws of 3000 and 10000 Da uses a passage route through the epithelium that changes with the waterllux and since they are too large to pass transcellu- lary through aqueous pores in the apical enterocyte-mem- branes, they probably pass via the paracellular route The dex- trans with M,:s of 40000 and 70000 Da use a route independent of waterflux, probably transcellularly via endo- cytosis by the enterocytes.

References

N. Pantzar, B.R. Westriim, A. Luts, S. Lundin, Regional small-intestinal permeability in vitro to different-sized dextrans and proteins in the rat, Stand. J. Gastroenterol., 1993, in press. T.Y. Ma, D. Hollander, R.A. Erickson, H. Troung, P. Krugliak, Is the small intestinal epithelium truly ‘tight’ to insulin permeation? Am. J. Physiol., 260 ( 199 1): G669- G676. G.M. Grass, S.A. Sweetana, In vitro measurements of gastrointestinal tissue permeability using a new diffusion cell, Pharm. Res., 5 ( 1988) 45-52.

F7 Metabolism of vasopressin, oxytocin and their analogues by juice and brush border membranes form the human gas- trointestinal (GI) tract. C. Soderberg-Ahlm’, A. Fjellestad- Paulsenb and S. Lundinb, “Ferring Pharmaceuticals, S-200 61 Malmii, Sweden; bDepartment of Clinical Pharmacology, Lund University Hospital, S-221 85 Lund, Sweden

The fact that biologically active peptides as large as nona- peptides can be absorbed from the GI-tract is very attractive from a therapeutic point of view. However, we know that the bioavailability of these substances is very low, and there is insufficient information on their metabolism in the human GI-tract.

The aim of the present study was to investigate the stability of the neurohypophyseal hormones arginine-vasopressin (AVP), oxytocin (OT) and their synthetic analogues in hu- man intestinal juice, small intestinal brush border mem- branes and gastric, rectal and colonic plasma membranes.

The gastrointestinal juice was collected from healthy male volunteers from different parts of the GI-tract (stomach, duodenum, distal jejunum, distal ileum) and was, after cen- trifugation, frozen and stored at -20°C. Peptides were in- cubated in undiluted juice at 37°C to a final peptide concen- tration of about 0.5 mM and the extent of degradation was determined by HPLC.

The brush border and plasma membranes were isolated from intestinal epithelium collected in patients undergoing surgery for localized tumors. Tissue was obtained from stom- ach, proximal jejunum, distal ileum, proximal colon, and rectum and was stored at - 70°C. Human intestinal micro- villi brush border membranes were prepared according to the

method described by Booth and Kenny [ 11. The incubation mixture consisted of l-10 ng membrane protein and 10v4 M peptide in a final volume of 100 ,~l with and without gluta- thion ( 10m4 M). The incubations proceeded at 37°C and degradation of the peptides was analysed by HPLC.

Depending on the local pH in the small intestine, degra- dation of 1 -deamino-8-D-arginine-vasopressin (DDAVP) might occur at various sites. This indicates an enzyme-de- pendent degradation that could be inhibited by adding a pro- teinase inhibitor (aprotinin). Degradation of DDAVP is rel- atively slow compared to AVP in ileal juice and the molecule is stable when exposed to mucosal intestinal membrane en- zyme activities. AVP and OT were also found to be rather slowly degraded by intestinal microvilli membranes and gas- tric, colonic and rectal plasma membranes.

When comparing the stability between different OT ana- logues in intestinal juice, C-terminal amides seem to be more stable than acids. Modifications made in the ring structure of the OT-molecule, substitution of L-Tyr with o-Tyr(Et) at position 2 increased the resistance to enzymatic attack.

In conclusion, the major enzymatic barrier to intestinal ab- sorption of OT, AVP and their analogues is present in the intestinal juice and not in the mucosa which, however, con- stitutes a major physical barrier to peptide transport.

References

1 A.G. Booth, A.J. Kenny, A rapid method for the prepa- ration of microvilli from rabbit kidney. Biochem. J. 142 (1974) 575-581.

PS Two derivatives of poly(acrylic acid) are able to inhibit trypsin activity. H.L. LueBen”, J.C. VerhoeQ, C.-M. LehP, A.G. de Bee? and H.E. Junginger”, Leiden/Amsterdam Cen- terfor Drug Research, Divisions of “Pharmaceutical Technol- ogy and bPharmacology, Center for Bio-Pharmaceutical Sci- ences, Leiden University, P. 0. Box 9502,230O RA Leiden, The Netherlands

It has been shown that Polycarbophil, a weakly crosslinked copolymer of poly(acrylic acid) derivatives, has a beniftcial influence on intestinal peptide absorption. This has been demonstrated using DGAVP (9-desglycinamide, 8-arginine vasopressin) by means of a fast releasing Polycarbophil for- mulation in vitro and in vivo (rat) [ 1,2]. There is evidence that Polycarbophil inhibits the activity of proteolytic en- zymes, which may result in an improved peroral peptide ab- sorption. Recent studies with N-a-benzoyl-L-arginine-ethyl- ester (BAEE), which was chosen as a standard substrate with well described kinetic properties, show a complete protection against degradation by the intestinal luminal enzyme trypsin under the influence of 0.35% Polycarbophil (pH 6.7) during 4 h in vitro. In contrast, no intact BAEE was found under the same conditions without using Polycarbophil after this pe- riod [3].

In the present study the effect of the non-crosslinked poly(acrylic acid) derivative Carbopol@ 934P was also in- vestigated. It was found that this polymer was also able to