Wortmannin, an Inhibitor of Phosphoinositide 3-Kinase, Inhibits Transcytosis in Polarized Epithelial Cells

doi:10.1074/jbc.270.47.28425

Wortmannin, an Inhibitor of Phosphoinositide 3-Kinase, Inhibits Transcytosis in Polarized Epithelial Cells (*)

From the Department of Pediatrics, Massachusetts General Hospital East, Charlestown, Massachusetts 02129

^§ To whom correspondence should be addressed:
Pediatric Gastroenterology, Massachusetts General Hospital East, 149 13th street Charlestown, MA 02129.
Tel.: 617-726-7991; Fax: 617-726-4172; hansens{at}helix.mgh.harvard.edu.

Abstract

Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits both basolateral to apical and apical to basolateral transcytosis of ricin in Fisher rat thyroid (FRT) cells by 50% at 100 nM in a continuous transcytosis assay. In MDCK cells, a similar effect of wortmannin on basolateral to apical transcytosis of ricin was found, whereas apical to basolateral transcytosis was inhibited to a lesser degree. Transcytosis of dimeric IgA in MDCK cells expressing the polymeric immunoglobulin receptor was also reduced to 50% of controls, suggesting that wortmannin inhibits membrane translocation rather than sorting of specific proteins in the transcytotic pathway. This effect of wortmannin is selective, however, in that endocytosis at the basolateral domain and recycling at both the basolateral and apical membrane domains are unaffected, and apical endocytosis and apical secretion are only moderately reduced. We have shown previously that cAMP stimulates a late stage in basolateral to apical transcytosis in MDCK cells through activation of protein kinase A (Hansen, S. H., and Casanova, J. E.(1994) J. Cell Biol. 126, 677-687). Elevation of cellular cAMP still induced a 100% increase in transcytosis in wortmannin-treated cells, but transcytosis was no longer increased when compared to cells which received no drugs. In contrast, in experiments using a 17°C block to accumulate ricin internalized from the basolateral surface in the apical compartment of MDCK cells, wortmannin had little effect on the stimulation of transcytosis by activators of protein kinase A observed under these conditions. The data thus suggest the existence of a wortmannin-sensitive step in the transcytotic pathway, positioned after endocytosis but prior to translocation into the protein kinase A-sensitive apical compartment, implying a role for phosphoinositide 3-kinase in an intermediate step in transcytosis in polarized epithelial cells.

Footnotes

↵* This work was supported by a Senior Research Fellowship from the Danish Cancer Society (to S. H. H.), by grants from Fabrikant Einar Willumsens Mindelegat, Dagmar Marshalls Fond, Martha Margrethe og Christian Hermansens Legat (to S. H. H.), and National Institutes of Health Grants NIH AI32291 and NIH DK33506 (to J. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽⁾ The abbreviations used are:

PI3K

phoshoinositide 3-kinase (uses PtdIns, PtdIns(4)P, and PtdIns(4,5)P as substrate)

pIgR

polymeric immunoglobulin receptor

PtdIns

phosphatidylinositol

PtdIns 3-kinase

phosphatidylinositol-specific 3-kinase (uses PtdIns as substrate)

8-Br-cAMP

cyclic 8-bromoadenosine 3′:5′-monophosphate

dIgA

dimeric IgA

PKA

protein kinase A

BSA

bovine serum albumin

TGN

trans-Golgi network

FSK

forskolin

Ro 20-1724

4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone.
- Received July 17, 1995.
- Revision received September 15, 1995.