Regulation of the Na, K‐ATPase activity of Madin‐Darby Canine Kidney cells in defined medium by Prostaglandin E₁ and 8‐bromocyclic AMP

Abstract

The role of PGE1 in regulating the activity of the Na+, K(+)-ATPase in Madin Darby Canine Kidney (MDCK) cells has been examined. PGE1 increased the initial rate of ouabain-sensitive Rb+ uptake by MDCK cells, a process that continued to occur over a 5-day period. The increase in the initial rate of ouabain-sensitive Rb+ uptake in MDCK cells treated with PGE1 could be explained by a 1.6-fold increase in the Vmax for ouabain-sensitive Rb+ uptake. The increase in the Vmax for ouabain-sensitive Rb+ uptake observed in MDCK cells under these conditions can be explained either by an increase in the number of active Na+ pumps, or by an increase in the efficiency of the Na+ pumps. Consistent with the former possibility is the observed increase in the number of ouabain binding sites, as well as the increase in Na+, K(+)-ATPase activity in cell lysates obtained from MDCK monolayers treated with PGE1. The involvement of cyclic AMP in mediating these effects of PGE1 on the Na+, K(+)-ATPase in MDCK cells is supported by: (1) the observation of similar effects in 8-bromocyclic AMP treated MDCK monolayers, and (2) a dramatic reduction of the stimulatory effects of PGE1 and 8-bromocyclic AMP on the Vmax for ouabain-sensitive Rb+ uptake, and on the number of ouabain binding sites in dibutyryl cyclic AMP resistant clone 3 (DBr3) (which is defective in cyclic AMP dependent protein kinase activity). PGE1 independent MDCK monolayers exhibit both an increase in the Vmax for ouabain-sensitive Rb+ uptake and an increase in the number of ouabain binding sites in response to 8-bromocyclic AMP. Apparently, the cyclic AMP phosphodiesterase defect in these PGE1 independent cells did not cause cellular cyclic AMP levels to be elevated to a sufficient extent to maximally increase the Na+, K(+)-ATPase activity in these variant cells.

Evidence for post-transcriptional regulation of Na,K-ATPase by prostaglandin E1

“…PGE 1 , a growth supplement in this medium, stimulates transport via the Na, K-ATPase, as well as growth [16,17]. Our investigations with variant MDCK cells indicate that the stimulatory effects of PGE 1 on the Na, K-ATPase occur by means of signaling pathways which are distinct from those responsible for the growth stimulatory effect of PGE 1 [1,17]. Indeed PGE 1 independent variants (PGE 1 Ind) of MDCK cells retain the stimulatory effect of PGE 1 on the Na, K-ATPase, while having lost the stimulatory effect of PGE 1 on growth [1,18,19].…”

“…Our investigations with variant MDCK cells indicate that the stimulatory effects of PGE 1 on the Na, K-ATPase occur by means of signaling pathways which are distinct from those responsible for the growth stimulatory effect of PGE 1 [1,17]. Indeed PGE 1 independent variants (PGE 1 Ind) of MDCK cells retain the stimulatory effect of PGE 1 on the Na, K-ATPase, while having lost the stimulatory effect of PGE 1 on growth [1,18,19]. The PGE 1 Ind MDCK cells had elevated intracellular cAMP levels, which can be attributed to their decreased cAMP phosphodiesterase activity [18].…”

“…Stock cultures of normal MDCK cells and DB r 3 cells were grown in DME/F12 supplemented with 5 μg/ml bovine insulin, 5 μg/ml human transferrin, 5 x 10 −12 M triiodothyronine (T 3 ), 5 x 10 −8 M hydrocortisone, 25 ng/ml PGE 1 , and 5 x 10 −8 M selenium (Medium K-1) in a humidified 5% CO 2 /95% air environment at 37°C. Stock cultures of PGE 1 Independent Clone I (Ind Cl I) were cultured in Medium K-1 lacking PGE 1 . MDCK cells were routinely subcultured using 0.53 mM EDTA and 0.05% trypsin in Phosphate-Buffered Saline (PBS).…”

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AbstractThe stimulatory effect of PGE 1 on the activity of the Na, K-ATPase in MDCK cells is associated with an increase in the rate of transcription of the Na, K-ATPase β1 subunit gene, and an increase in the rate of biosynthesis of the Na, K-ATPase [1]. In order to further define the molecular mechanisms, transient transfection and biosynthesis studies were conducted with Dibutyryl cAMP resistant (DB r ) MDCK cells, defective in cAMP dependent protein kinase, and PGE 1 Independent (PGE 1 Ind) MDCK cells with elevated intracellular cAMP.

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Involvement of EP1 and EP2 receptors in the regulation of the Na,K-ATPase by prostaglandins in MDCK cells

“…Previously, we reported that PGE 1 , and 8-Bromocyclic AMP (8Br-cAMP) cause an increase in the Na,K-ATPase activity of MDCK cells [19], which was associated with an increase in the level of the Na,K-ATPase in this cell type [4]. The Na,K-ATPase is a protein which is composed of an α subunit with catalytic activity, as well as a β subunit, which facilitates the integration of the protein into the plasma membrane [20].…”