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Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase
Abstract:
Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Prostaglandin E1 (PGE1) stimulates Madin Darby Canine Kidney (MDCK) cell growth and differentiation. Variants resistant to cyclic AMP showed reduced protein kinase activity but retained growth responses to PGE1, indicating separate mechanisms for growth and differentiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Prostaglandin E1 (PGE1) influences Madin Darby Canine Kidney (MDCK) cell growth and differentiation.
- PGE1's effects on MDCK cell growth and differentiated functions, like dome formation, occur under different conditions (subconfluent vs. confluent).
- Separate mechanisms may underlie PGE1's influence on MDCK cell growth and differentiation.
Purpose of the Study:
- To investigate if PGE1 affects MDCK cell growth and differentiation through distinct pathways.
- To characterize the role of cyclic AMP-dependent protein kinase in mediating PGE1's effects.
- To analyze the functional and growth responses of cyclic AMP-resistant MDCK variants to PGE1.
Main Methods:
- Selection and characterization of dibutyryl cyclic AMP resistant MDCK variants.
- Assay of cyclic AMP-dependent protein kinase and phosphodiesterase activities in variant and normal MDCK cells.
- Evaluation of PGE1-induced growth and dome formation in variant MDCK cells.
- Measurement of Na+/K+-ATPase activity via ouabain-sensitive Rb+ uptake.
Main Results:
- Dibutyryl cyclic AMP resistant variants exhibited reduced cyclic AMP-dependent protein kinase activity.
- These variants maintained normal growth responses to PGE1.
- PGE1-induced dome formation was significantly reduced in variants, although other inducers were still effective.
- PGE1 elevated Na+/K+-ATPase activity in confluent MDCK cell monolayers.
Conclusions:
- PGE1 likely impacts MDCK cell growth and differentiation via separate signaling pathways.
- Reduced cyclic AMP-dependent protein kinase activity in variants correlates with impaired PGE1-induced differentiation.
- The Na+/K+-ATPase is implicated as a functional parameter affected by PGE1 during MDCK cell differentiation.