ATP stimulates Cl⁻ secretion and reduces amiloride‐sensitive Na⁺ absorption in M‐1 mouse cortical collecting duct cells

Abstract

Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M‐1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride‐sensitive ISC component. The ATP‐induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl− and in the presence of the apical Cl− channel blockers diphenylamine‐2‐carboxylic acid (DPC, 1 mM), DIDS (300 μM) and niflumic acid (100 μM). The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 μM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. ATP and UTP applied to either the apical or the basolateral bath equi‐potently stimulated ISC while ‘purified’ ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT‐PCR. Intracellular calcium concentration ([Ca2+]i) measurements using fura‐2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 μM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre‐incubated with the calcium chelator BAPTA AM. We conclude that in M‐1 cells extracellular ATP reduces amiloride‐sensitive Na+ absorption and stimulates Cl− secretion via calcium‐activated Cl− channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane.

Sodium-Dependent Regulation of Renal Amiloride-Sensitive Currents by Apical P2 Receptors

“…Positive immunofluorescence was also seen for ␣-, ␤-, and ␥-ENaC subunits in the CD of both Na ϩ -replete and Na ϩ -restricted rats; this immunofluorescence appeared stronger in the PC of Na ϩ -restricted rats. With respect to PC only, in Na ϩ -replete rats strong immunostaining for P2X 4 and 6 and for P2Y 4 , 6 , and 11 was consistently observed in the apical domain of PC throughout the CD, and weaker apical staining was also seen for P2X 2 and P2Y 2 (both localized to the medullary CD only) and ␣-ENaC. Strong basolateral immunostaining for P2X 4 and 6 was seen in all PC; basolateral staining for P2Y 2 was evident only in medullary CD PC.…”

“…It has already been established that the activation of apically expressed P2R by extracellular nucleotides can decrease I am-s amplitude in the CD, [11][12][13][14][17][18][19] but argument remains over the P2R subtype(s) responsible. Previous pharmacologic profiling studies using mouse isolated CD reported that apical G protein-coupled P2Y 2 -like receptors mediated ATP-evoked inhibition of ENaC activity.…”

“…9,10 Given the importance of ENaC in controlling arterial BP, it is perhaps not surprising that there have been several studies on the modulation of its activity by P2R in the CD. To date, P2R-mediated modulation of ENaC activity has been demonstrated by means of electrophysiologic techniques in murine and amphibian immortalized renal CD/distal nephron cell lines 7,8,[11][12][13][14][15] and in the Xenopus oocyte heterologous expression system (using recombinant rat P2XR and ENaC proteins). 16 It has also been reported in the CD of rat 17 and mouse 18,19 using in vivo and in vitro luminal perfusion studies, respectively.…”

ENaC, renal sodium excretion and extracellular ATP

“…In turn, raised [Ca 2+ ] int resulted in the activation of PKC which was proposed to mediate the inhibition of ENaC in rabbit CD cells. UTP inhibition of sodium transport was also confirmed in the mouse M1 collecting duct (CD) cell line [31] and in mouseisolated CDs [32]. Curiously, the early data of Cuffe et al…”

Cl- and regulation of pH by MDCK-C11 cells

“…receptors have been detected in C11 cells (22). In addition, it was shown that extracellular ATP stimulates Cl -secretion while increasing cell Ca 2+ in both renal and digestive epithelial cells (20,23). The activation of Clconductance may modulate H + -ATPase in C11 cells.…”