Coupled ATP and potassium efflux from intercalated cells

Abstract

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (τ; 10 dynes/cm(2)) -induced, BK-dependent, K efflux. We found that 10 μM ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P(2) receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited τ-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-β4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and τ-induced K efflux. Similarly, carbenoxolone (25 μM), which blocks connexins, putative ATP pathways, blocked τ-induced K efflux and ATP secretion. Compared with BK-β4(-/-) mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for τ-induced ATP release in IC.

Fig. 1.

P₂ signaling in C11. A: plot of intracellular Ca²⁺ concentration ([Ca²⁺]_i) obtained from fura 2 imaging of C11 exposed to ATP in the presence or absence of 1 mM extracellular calcium. B: plot of [Ca²⁺]_i of C11 preincubated with suramin and exposed to ATP in the presence of extracellular calcium and ionomycin. C: summary bar plot of average peak [Ca²⁺]_i for each condition in A and B. *Significant (P < 0.005) difference from baseline. †Significant difference (P < 0.005) from suramin.

Fig. 2.

Flow-induced ATP efflux in C7 and C11. ATP secreted from C7 and C11 under static (control) and flow (10 dynes/cm²) conditions. Values are means ± SE. *Significant (P < 0.005) difference from static. †Significant (P < 0.005) difference between cell types.

Fig. 3.

Effects of large-conductance, calcium-activated K channel subunit BK-β4 small interference (si) RNA on ATP secretion in C11. A: Western blot analysis of protein from C11 that were untreated (C11), transfected with BK-β4 siRNA (siRNA BK-β4) or nontarget siRNA (siRNA control). B: BK-β4 siRNA silenced ∼82% of BK-β4 compared with normal conditions (P < 0.02; n = 6). C: flow-induced ATP release was blocked by BK-β4 siRNA (n = 7), but not by siRNA control (n = 7).

Fig. 4.

Effects of suramin on flow-induced K efflux from C11 (n = 6). Symbols are the same as for Fig. 2.

Fig. 5.

ATP activation of BK-α/β4-mediated K efflux. A: under static conditions, the addition of ATP decreased C11 intracellular K/DNA content; this effect was blocked by suramin and paxilline (n = 5). B: ATP-induced K efflux was significantly decreased by siRNA BK-β4, but not by siRNA control (n = 4). *Significant (P < 0.02) difference from control.

Fig. 6.

Effects of ATP on BK-dependent cell volume decrease. A: representative images of calcein intensity of C11 before and after the addition of 10 μM ATP. The significant increase in calcein intensity indicated a decrease in cell volume compared with control (P < 0.01; n = 8). B: summary plot of calcein intensity before (control) and after ATP. *Significant difference from control.

Fig. 7.

Effects of carbenoxolone on ATP and K secretion. Carbenoxolone blocks shear stress (τ)-induced ATP efflux (A) and intracellular ATP content (B). C: carbenoxolone block of τ-induced release of intracellular K (n = 6). Symbols are the same as for Fig. 2.

Fig. 8.

Summary bar plots of urinary ATP secretion for wild-type (WT) and BK-β4^−/− mice on control and high-K (HK) diets. *Significant (P < 0.005) difference from control. †Significant (P < 0.005) difference from WT.