Effect of cholera toxin, oxygen and tissue submersion on prorenin and HCG secretion from human villous placental tissue in vitro

Abstract

EFFECT OF CHOLERA TOXIN, OXYGEN AND TISSUE SUBMERSION ON PRORENIN AND HCG SECRETION FROM HUMAN VILLOUS PLACENTAL TISSUE IN VITRO. A.M. Poisner, G.J. Downing and R. Poisner, Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS USA. Renin, mostly as prorenin (PR), is present in extremely low concentrations in the villous placenta. We now report that modified organ cultures of villous placenta, incubated for periods up to 72 hrs, show an increase in PR secretion and that this is greatly enhanced by treatment with cholera toxin (CTX), oxygen and tissue elevation. 1-3 mm pieces of villous placenta were placed in 24-well tissue culture plates and incubated in 2 ml of modified CMRL medium (either submerged or placed upon filters at the gas-liquid interface) under 5% CO 2 (in air or in 95% 02). PR was released at very low concentrations and this was greatly increased by CTX (100 ng/ml) and by 95% 02. PR secretion (uU/mg protein) increased with time. At 72 hrs, CTX increa/sed PR from 109 • 38 to 19,450 • 4,860 in air; and from 1,640 • 367 to 108,000 = 14,900 in 02. HCG secretion (ng/mg protein) was not stimulated in air (from 74.9 • 8.4 to 95.2 • 19.7) but was stimulated in O 2 (from 88.0 • 14.8 to 629 • 119). Elevation of the placental pieces enhanced the release of PR (20 fold in air; and 8 fold in Oz). There was a negligible effect on HCG secretion. The effects on placental PR secretion are far greater than any previously reported. These studies show differences in PR and HCG regulation and describe a new model for study of placental renin secretion. A.59 LONG-TERN ORGANOTYPIC CULTURE OF HUHAN PLACENTAL EXPLANTS: HORHONAL AND |Mt4UNOCYTOCHENICAL EVALUATIGN OF THE EXPERIMENTAL 14OOEL. 8rurm PotL iot t i 1, Scott Keesting 1, Mei Zhang 2, Eva Back61, Joseph Cabs 1, Francis Lee 1, David Schwartz 2, Naurice Psniget 1 and Andr~ Nahmias 1,2, Departments of Pediatr ics 1 and Pathology 2, Emery Universi ty, At lanta, CA, USA. The ef fects of d i f fe ren t infect ious agents on the various types of placental ce l ls require long-term ex vivo maintenance of placental tissue, pa r t i cu la r l y in case of viruses such as HIV and CNV = ui th long repl icat ion cycles. For th is purpose, we have studied several variables in placental t issue explants obtained a f ter cesarean section and incubated in RPH! supplemented with 104 FCS, with or without an t i b io t i c , for periods up to 13 days. The medium yes replaced every day and samples of the supernatsnt were frozen for later test ing of hPL release and glucose consumption. The t issue was also f ixed end embedded st d i f f e ren t periods for microscopic examinations. During the incubation period (11 to 13 days), the pH, pO 2 and 10(;02 were Bonitored and found to remain constant. The glucose u t i l i z a t i o n represented 25~ of the i n i t i a l concentration (1.74 g / l ) in the medium and was found to be the same for a l l the incubation in terva ls . Af ter a rapid decrease, hPL in the supernatant reached a basal level (2.77=1.51 ng/ml/mg/Z4hr.), which remained stable for mere than 10 days. Af ter 13 days, the chorlonir v i l l i trophobtast was s t i l t h i s to log ica l l y recognizable as a dist inguishable layer with an apical brush border. Dense secretory granules of hPL, characterized by i ~ c y t o c h e m i s t r y , were local ized in the syncytiotrophoblast layer. P ro l i fe ra t ion marker (Ki67), detected by the HIB1 monoctona{ antibody, was also observed in some residual cytotrophoblast ce l ls . At the end of the cul ture, blood capi l la r ies were no longer observed in the connective t issue stroma. Long-term organotypic cultures should help to understand better the pathogenesis of infect ious agents in the placenta, in the present model, the placental t issue can be incubated ex vivo for prolonged periods, without necessari ly the addit ion of any an t ib io t i c which could in ter fe re in studies of some infect ious agents. Horeover, unl ike many other orgsnotypic cul ture procedures, th is method allows extensive washing of the explant in order to el iminate the remaining infect ious inocutum and to measure the release of placental products in cul ture supernatants.