Abstract
The effect and mechanism of action of glucocorticoids (GC) on Na-Pi cotransport were evaluated in opossum kidney cells. Dexamethasone (1-1000 nM) inhibited sodium-dependent Pi uptake in a time- and concentration-dependent manner. Inhibition was maximal after a 6-h incubation with dexamethasone and was prevented by cycloheximide and actinomycin D. The effect was related to a 37% decrease of the Vmax value after incubation with 100 nM dexamethasone. The effect of dexamethasone was mimicked by cortisol and blocked by GC receptor antagonists RU38486 and progesterone. GC affected neither glucose or alanine uptake nor Na/H exchange activity. Inhibition of Pi uptake persisted when Na/H was blocked by amiloride or dimethylamiloride. GC had no effect on basal or parathyroid hormone- and forskolin-stimulated intracellular cAMP content. Dexamethasone and extracellular cAMP, parathyroid hormone, or 3-isobutyl-1-methylxanthine had additive inhibitory effects on Pi uptake. Staurosporine, GF109203X, or calphostin C (three dissimilar inhibitors of protein kinase C (PKC)) and PKC down-regulation blunted the inhibitory effect of glucocorticoids on Pi uptake. GC increased both membrane-bound PKC activity and the membrane/cytosol PKC activity ratio. This is the first report of GC activation of PKC in renal cells, which appears to mediate the steroid inhibitory effect on Pi transport.
Highlights
From the $Department of Physiology and Institut National de la Santeet de la Recherche Medicale (INSERM) U 251, Faculte de Mkdecine Xauier-Bichat, Uniuersite'Denis-Diderot-Paris 7, F-75018, Paris,the Waboratoire de Biochimie-Biologie Cellulaire, INSERM U 181, Faculte de Medecine Saint-Antoine, 75571, Paris, and the Jbaboratoire d'Endocrinologie Expirimentale, Centre Oscar Lambret, 59000 Lille, France
In order to ascertain whether theinhibitory effect of dexamethasone on Pi uptake was not related to dexamethasonemediated inhibition of phosphodiesterase activity resulting in an increase in CAMPlevel potentially leading to PKA activation, Pi uptake was measured in thperesence of IBMX
In order to correlate this result with the GC effect on Na-Picotransport, PKC activity was measured in thepresence of 10PM cycloheximidein control and dexamethasone-treated cells
Summary
Materials-Insulintr,ansferrinh,ydrocortisoned,examethasone, progesterone,aldosterone, methyl-u-~-g~ucopyranosid(Me GP), L-alanine, sodium selenite, phenylmethylsulfonyl fluoride, benzamidine, leupeptin, cycloheximide, forskolin, adenosine 3',5'-monophosphate, actinomycin D, phorbol 12-myristate 13-acetate(PMA), fragment41-34) of bovine PTH, amiloride, and 3-isobutyl-1-methylxanthine(IBMX) were purchasedfromSigma. RU38486 and GF109203X werekindly provided, respectively, by Roussel-Uclaf (Romainville, France) and Glaxo. R 59022 was from Janssen Life Science products (Olen, Belgium). Dimethylamiloride (DMA) was a generous gift from C. Frelin (Centre de biochimie, Centre National de la Recherche Scientifique, Universite de Nice, France). Tracers were from the following sources: carrier-free NalZ51,K,H3*P0,, ~-[2,3-~Hlalanine, a2n2dNaC1were from Amersham (Amersham, UnitedKingdom), and [Y-~~PIATP maenthdylc~-~-[U-'~C]glucopyranosi(d[le4C1MGP)were from DuPontNEN Research Products (Les Ulis, France). Cell Culture-OK cells (passages 100-108) were grown toconfluence i n 24-well trays ina medium consistingof a 1:l (v/v) mixtureof Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 15
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