Loss of the PGE₁ requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Abstract

Prostaglandin E1(PGE1), one of the components in the hormone‐supplemented, serum‐free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K‐1), is required for both long‐term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE1, requirement for long‐term growth in Medium K‐1. These variants will be useful in identifying the molecular events initiated by PGE1 which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE1 independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K‐1 and in serum‐supplemented medium; the growth rate was lower in Medium K‐1 lacking PGE1. In contrast, PGE1 independent clone 1 grew at an equivalent rate in Medium K‐1 minus PGE1, and in serum‐supplemented medium. When PGE1 was added to K‐1 minus PGE1, less growth of PGE1 independent clone 1 was observed. A similar observation was made with one other PGE1 independent clone which was studied. A hormone deletion study indicated that PGE1 independent clone 1 still retained growth responses to the other four supplements in Medium K‐1 (insulin, transferrin, T3, and hydrocortisone). The molecular alterations associated with loss of the PGE1 requirement for long‐term growth were examined. At confluency, all of the PGE1 independent clones studied had higher intracellular cyclic AMP levels following PGE1 treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE2 for the PGE1 receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE1 independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE1 on dome formation by the variant cells was also examined. The frequency of dome formation by PGE1 independent clone 1 was enhanced in a dosage‐dependent manner, like normal MDCK cells. This observation suggests that PGE1 affects MDCK cell growth and dome formation by different mechanisms.

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

“…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].…”

“…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]. In contrast Dibutyryl cAMP resistant (DB r ) MDCK cells retain the stimulatory effect of PGE 1 on growth, but have lost the stimulatory effect of PGE 1 on the Na, K-ATPase [17].…”

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

“…Previously, the results of in situ hybridization have indicated that EP1 receptor mRNA is found primarily in the collecting duct [43,44], while EP 2 receptors are present predominantly in distal tubule cells as well as glomeruli [7,30]. Of particular interest in these regards, targeted disruption of EP2 was observed to cause saltsensitive hypertension, supporting a role for EP2 in renal salt handling [44,45].…”

“…In this report the reduction in Na,KATPase activity following a 10 min incubation, was presumably due to a decrease in the level of the Na,K-ATPase in the plasma membrane, rather than to changes in overall gene expression. Such acute effects of PGE 1 and PGE 2 may possibly be mediated by EP3 receptors (which are localized in the collecting duct) [43,44,50,51]. However, the inhibitory effect of PGE 2 on Na + absorption in the collecting duct has been shown to occur via a Ca 2+ dependent mechanism, and was not prevented by a potent EP3 agonist [44,51].…”

“…Such acute effects of PGE 1 and PGE 2 may possibly be mediated by EP3 receptors (which are localized in the collecting duct) [43,44,50,51]. However, the inhibitory effect of PGE 2 on Na + absorption in the collecting duct has been shown to occur via a Ca 2+ dependent mechanism, and was not prevented by a potent EP3 agonist [44,51]. Nonsteroidal anti-inflamatory drugs (NSAIDs) enhance the urinary concentrating ability of the kidney by preventing such inhibitory effects of PGE 2 in the collecting duct.…”