| Abstract |
To facilitate investigation of Ca2+ and Na+ transport by parathyroid
hormone (PTH)-sensitive distal convoluted tubules (DCT), we established a
stable line of these cells. Hybrid primary cultures consisting of DCT and
cortical ascending limb cells were isolated from mice by immunodissection.
Cells were infected with chimeric adenovirus 12-Simian virus 40 and grown
in DMEM/Ham's F-12 media until mock infectants had expired. Cells were
then passaged and cloned by limiting dilution. We measured ouabain (OUA)-
suppressible oxygen consumption (QO2, nmol O2 consumed x min^-1 x mg prot^-1)
in clones exhibiting PTH-stimulated cAMP formation >= 5x control. As
shown in the table, in the mouse DCT (MDCT) cell line designated 210B3,
chlorothiazide (CTZ) and amiloride (AMIL) significantly inhibited Q02
(P<0.05); bumetanide (BUM) had no significant effect (either alone or
when added to CTZ). Further characterization of these cells was
accomplished by measuring 22Na+ and of 36Cl- uptake at an external NaCl
concentration of 140 mM. CTZ significantly decreased 22Na+ and 36Cl- uptake
by 36 +- 3% and 52 +- 4%, respectively (P<0.05). BUM had no effect. AMIL
decreased 22Na+ uptake by 40 +-4% (P<0.05), but had no effect on 36Cl-
uptake. CTZ and AMIL increased 45Ca2+ uptake by 37 +- 3% and 28 + 2% (P<0.05),
respectively, while BUM had no effect. Activation of adenylyl cyclase
by PTH, inhibition of Na+ and Cl- transport and stimulation of Ca2+
transport by thiazide diuretics, together with the lack of effect of BUM
is consistent with a DCT phenotype. In summary, the results demonstrate
the development of a cell line, MDCT 210B3, that is PTH sensitive,
expresses thiazide inhibitable Na-Cl cotransport, AMIL-sensitive Na+
uptake, and thiazide- and AMIL-stimulated Ca2+ uptake.
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