Effects of controlled ovarian stimulation on vascular barrier and endothelial glycocalyx: a pilot study.

PURPOSE: Controlled ovarian stimulation significantly amplifies the number of maturing and ovulated follicles as well as ovarian steroid production. The ovarian hyperstimulation syndrome (OHSS) increases capillary permeability and fluid extravasation. Vascular integrity intensely is regulated by an endothelial glycocalyx (EGX) and we have shown that ovulatory cycles are associated with shedding of EGX components. This study investigates if controlled ovarian stimulation impacts on the integrity of the endothelial glycocalyx as this might explain key pathomechanisms of the OHSS. METHODS: Serum levels of endothelial glycocalyx components of infertility patients (n=18) undergoing controlled ovarian stimulation were compared to a control group of healthy women with regular ovulatory cycles (n=17). RESULTS: Patients during luteal phases of controlled ovarian stimulation cycles as compared to normal ovulatory cycles showed significantly increased Syndecan-1 serum concentrations (12.6 ng/ml 6.1125th-19.1375th to 13.9 ng/ml 9.625th-28.975th; p=0.026), indicating shedding and degradation of the EGX. CONCLUSION: A shedding of EGX components during ovarian stimulation has not yet been described. Our study suggests that ovarian stimulation may affect the integrity of the endothelial surface layer and increasing vascular permeability. This could explain key features of the OHSS and provide new ways of prevention of this serious condition of assisted reproduction.

Differential inhibitory effects of serine/threonine phosphatase inhibitors and a calmodulin antagonist on phosphoinositol/calcium- and cyclic adenosine monophosphate-mediated pancreatic amylase secretion.

BACKGROUND: Protein phosphorylation and dephosphorylation events are considered to be key steps in the control of agonist-induced pancreatic enzyme release. This study was designed to characterize the role of serine/threonine phosphatases in phosphoinositol/calcium- and cyclic adenosine monophosphate (cAMP)-mediated stimulus-secretion coupling in rat pancreatic acini. METHODS: Isolated rat pancreatic acini were incubated with either the serine/threonine phosphatase inhibitors okadaic acid, calyculin A, and cyclosporin A or the calmodulin antagonist W-7. Amylase secretion was stimulated with cholecystokinin (CCK)-8, secretin, vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP), and the intracellular second messengers calcium and cAMP were determined. RESULTS: Okadaic acid or calyculin A reduced secretagogue-stimulated amylase release to near-basal levels. Inhibition of cAMP-mediated secretion (by VIP, secretin, or PACAP) occurred at lower concentrations than with inositol triphosphate (IP3)/Ca(2+)-dependent enzyme release (via CCK). Cyclosporin A diminished CCK-8-stimulated secretion by 35%, whereas secretion in response to cAMP-mediated secretagogues was not affected. W-7 completely inhibited acinar secretion in response to cAMP-or IP3/Ca(2+)-mediated secretagogues. Binding of 125I-CCK-8- or 125I-PACAP-(1-27) to acini was not influenced by the phosphatase inhibitors or W-7. Okadaic acid and calyculin A affected neither CCK-8-stimulated intracellular Ca2+ release nor PACAP-(1-27)-stimulated cAMP synthesis, whereas W-7 inhibited by 50% and 40%, respectively. CONCLUSIONS: The inhibitory profiles of okadaic acid, calyculin A, cyclosporin A, and W-7 indicate that phosphatases 1 and 2A play a relevant role in cAMP-mediated enzyme release, whereas phosphatases 1 and 2B are predominantly involved in IP3/Ca(2+)-dependent stimulus-secretion coupling. The calmodulin antagonist W-7 interferes at multiple steps of intracellular signal-transduction pathways.

Calyculin A, okadaic acid and W-7 interfere with a distal step in pancreatic acinar signal transduction.

The effects of the serine/threonine phosphatase inhibitors calyculin A, okadaic acid and the calmodulin antagonist W-7 on amylase secretion were studied in pancreatic acini. Calyculin A and okadaic acid dose-dependently inhibited amylase secretion to basal levels when stimulated with the intracellularly acting secretagogues thapsigargin, 8-br-cAMP or PMA. W-7 dose-dependently inhibited thapsigargin- or 8-br-cAMP-induced amylase secretion. In combination, thapsigargin, 8-br-cAMP and PMA induced amylase secretion comparable to the stimulation by cholecystokinin. Their effect was significantly inhibited by calyculin A, okadaic acid or W-7. These data imply that type 1- and 2b-phosphatases and calmodulin play a key role in the stimulation of exocrine pancreatic secretion at a distal step of both the Ca2+/IP3- and cAMP-mediated signal-transduction pathways.

Serine/threonine phosphatases play a role in stimulus-secretion coupling in pancreatic acinar cells.

The role of serine/threonine phosphatases in Ca2+/IP3- and cAMP- mediated stimulus-secretion coupling was investigated in isolated pancreatic acinar cells. Cyclosporine A, an inhibitor of type 2b serine/threonine phosphatases, maximally reduced CCK-8-stimulated amylase secretion by 33%. In contrast, the secretory response to secretin or PACAP-(1-27) was not significantly altered by cyclosporine A independent of the secretagogue-concentration okadaic acid significantly reduced amylase release, induced by Ca2+/IP3-mediated- (CCK-8) or cAMP-mediated agonists (secretin, PACAP-(1-27), VIP) at concentrations that primarily inactivate type 1 and 2b phosphatases. Calyculin A, another type 1 and 2a phosphatase inhibitor, had a similar inhibitory effect on CCK-8-, secretin- or PACAP-(1-27)-induced secretion. In permeabilized acini, cyclosporine A reduced calcium-induced amylase release by 20%, whereas okadaic acid and calyculin A had an inhibitory effect by 55% and 52%, respectively. The ultrastructure of CsA-incubated acinar cells was not different from vehicle-incubated control lobules. In contrast, incubation with okadaic acid for 60 min resulted in morphological alterations of the Golgi apparatus, leading to a fragmentation of Golgi cisternae into small vesicles. Our data suggest a role of type 1 and 2b phospatases in stimulus-secretion coupling of both signal-transduction pathways in pancreatic acinar cells. These phosphatases might also be important for the maintenance of pancreatic cellular ultrastructure.