Complete inhibition of Na+, K+, Cl− cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase

Abstract

This study examines the involvement of hormones and neuromediators in the regulation of Na+, K+, Cl- cotransport in renal epithelial cells using Madin-Darby canine kidney cells with low transepithelial electrical resistance (194+/-47 Omega/cm2). In this cell line, Na+, K+, Cl- cotransport measured as bumetanide-sensitive 86Rb influx was inhibited up to 50-60% with agonists of P2-purinoceptors (ATP approximately ADP>UTP>AMP), slightly (15-30%) increased by activators of cAMP signaling (forskolin, 8-Br-cAMP) and was insensitive to activators of cGMP signaling (8-Br-cGMP, nitroprusside), EGF, angiotensin II, bradykinin, methacholine, propranolol, vasopressin, adenosine, dopamine and histamine. Thirty min of preincubation of MDCK cells with 0.1 microM PMA completely blocked the activity of Na+, K+, Cl- cotransport whereas down-regulation of this enzyme by 24 h of preincubation with 1 microM PMA activated Na+, K+, Cl- cotransport by 60% and abolished the effect of short-term treatment with PMA. Regulation of Na+, K+, Cl- cotransport by ATP was insensitive to down-regulation of PMA-sensitive isoforms of protein kinase C. In addition, an inhibitor of protein kinase activity, staurosporine, abolished the effect of 0.1 microM PMA but did not change inhibition of this carrier by ATP. Thus, these results show for the first time that P2-purinoceptors and PMA-sensitive isoforms of protein kinase C play a key role in the regulation of Na+, K+, Cl- cotransport in MDCK cells. These results also show that neither PMA- nor staurosporine-sensitive forms of protein kinase are involved in the inhibition of Na+, K+, Cl- cotransport by activators of P2-purinoceptors.

Purinergic Modulation of Na + ,K + ,Cl − Cotransport and MAP Kinases is Limited to C11-MDCK Cells Resembling Intercalated Cells from Collecting Ducts

“…We have reported previously that the addition of PMA and ATP slightly potentiated the activity of Na + ,K + pump in MDCK cells, whereas Na + ,K + ,Cl − cotransport was completely inhibited by PMA, partially inhibited (∼40-50%) by ATP and augmented by 30% under activation of adenylate cyclase with forskolin (Gagnon et al, 1998). Data obtained on MDCK cells in the present study confirmed these findings (Tables 2 and 3).…”

“…The data obtained in the present study show that the recently discovered inhibition of Na + ,K + ,Cl − cotransport in MDCK cells by agonists of P 2Y receptors (Gagnon et al, 1998) is a feature of REC from CD. As with our findings in CD-derived MDCK cells, we noted that ATP inhibited Na + ,K + ,Cl − cotransport in REC from rabbit and rat CD but did not affect this carrier in cells from PT and DT (Table 3).…”

“…Protein content was measured by Lowry's method. As reported previously, the kinetics of 86 Rb uptake by MDCK cells were linear up to at least 20 min (Gagnon et al, 1998). Unless otherwise indicated, an incubation time of 15 min was used to determine the initial rate of K + influx.…”

“…We reported earlier that in MDCK cells, Na + ,K + ,Cl − cotransport is insensitive to agonists of P 1 -purinergic, ␣-adrenergic, cholinergic and dopaminergic receptors as well as to vasopressin, bradykinin, angiotensin II and 8-Br-cGMP, but is completely blocked by activation of protein kinase C (PKC) with 4␤-phorbol 12-myristate 13-acetate (PMA) and is partially inhibited by extracellular ATP (Gagnon et al, 1998). Recently, we demonstrated that ATP-induced inhibition of Na + ,K + ,Cl − cotransport in MDCK cells is triggered by P 2Y purinoceptors (Gagnon et al, 1999a).…”

Transient Activation and Delayed Inhibition of Na+,K+,Cl–Cotransport in ATP-treated C11-MDCK Cells Involve Distinct P2Y Receptor Subtypes and Signaling Mechanisms

“…We also demonstrated the lack of effect in the sustained inhibition of NKCC of several other intermediates of P2Y-induced signaling in MDCK cells (e.g. phospholipases C and A 2 , protein kinases A, C, and extracellular signal-regulated kinase 1/2 (ERK1/2)) (20,21,24). However, sustained application of ATP to C11, but not to C7, cells can activate the stress-sensitive protein kinase c-Jun N-terminal kinase 1 (JNK1) (24).…”