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Department of Medicine, University of British Columbia, Vancouver Hospital and Health Sciences Centre, Koerner Pavilion, Vancouver, British Columbia, Canada V6T 1Z3
Prostaglandins
have diverse effects on renal electrolyte reabsorption, inhibiting NaCl
absorption in the thick ascending limb and modulating sodium and
calcium transport in cortical collecting cells. It is unclear what
effect, if any, prostaglandins have on tubular magnesium handling. The
effects of prostaglandin E2 (PGE2) were studied on
immortalized mouse distal convoluted tubule (MDCT) cells by measuring
cellular cAMP formation with radioimmunoassays and
Mg2+ uptake with fluorescence
techniques. Intracellular free
Mg2+ concentration
([Mg2+]i)
was measured on single MDCT cells using microfluorescence with mag-fura
2. To assess Mg2+ uptake, MDCT
cells were first Mg2+ depleted to
0.22 ± 0.01 mM by culturing in
Mg2+-free media for 16 h and then
placed in 1.5 mM MgCl2, and the changes in
[Mg2+]i
were determined.
[Mg2+]i
returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate,
d([Mg2+]i)/dt,
of 173 ± 8 nM/s. Indomethacin, 5 µM, diminished basal Mg2+ uptake, suggesting that
endogenous prostaglandins may stimulate Mg2+ entry in control cells.
PGE2 stimulated
Mg2+ entry in a
concentration-dependent manner with maximal response of 311 ± 12 nM/s, at a concentration of
10
7 M, which represented an
80 ± 3% increase in uptake rate above control values. This was
associated with a sixfold increase in intracellular cAMP generation.
PGE2-stimulated
Mg2+ uptake was completely
inhibited with the Rp diastereoisomer of adenosine
3',5'-cyclic monophosphothionate (Rp-cAMPS), a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and
partially by chelerythrine, a protein kinase C inhibitor. Accordingly,
PGE2-mediated
Mg2+ entry rates involve multiple
intracellular signaling pathways. These studies demonstrate that
PGE2 stimulates
Mg2+ uptake in a cell line of MDCT.
intracellular magnesium; fluorescence; intracellular adenosine 3',5'-cyclic monophosphate; prostaglandin E2; indomethacin
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