Cellular mechanisms of ATP‐induced hyperpolarization in renal epitheloid MDCK‐cells

Abstract

Previous studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)-cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoyl phorbol 13-acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5-trisphosphate (IP3), inositol 1,3,4,5-tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the cell membrane.

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

“…Transient elevation of [Ca 2ϩ ] i during 2-5 min of ATP addition has been documented in different subtypes of MDCK cells (20,34,35), including C11 cells (24). We observed that [Ca 2ϩ ] i response in C11 cells treated with ATP in medium A was slightly attenuated in the absence of extracellular Ca 2ϩ but was almost completely abolished in cells loaded with the intracellular Ca 2ϩ chelator BAPTA (Fig.…”

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

“…Two additional lines of evidence indicate that store-operated channels make, at most, a minor contribution to the RGD-induced influx. First, we found that the magnitude of [Ca21]i transients resulting from ATP stimulation is similar in the presence or absence of external calcium, suggesting that, although it is substantial in other clones (Paulmichl et al, 1990), stimulation of store release does not trigger a large store-operated influx component in the clone of cells we have used. Second, we can rule out the possibility that RGD beads induce a rapidly inactivating storeoperated Ca21 influx mechanism like that triggered by TG, because that influx was Ni2+-insensitive, unlike the RGD bead response, which is Ni2+-sensitive (Figure 8b).…”

“…Thus, in the present study we focused on mechanisms that elicit the initial sharp increase in [Ca2+]i (the RGDinduced [Ca2+]i "transient"). Both bead adhesion and calcium signaling are specific to RGD and are decreased by antibodies that inhibit the function of av133 and avP35 integrins (Sjaastad et al, 1994 We examined the dependence of (Paulmichl et al, 1990). Ca2+ release occurs because of activation of phospholipase C and generation of IP3 via a phorbol ester and pertussis toxin-sensitive mechanism (Paulmichl et al, 1990 , 1993), tyrosine phosphorylation-dependent activation of phospholipase C (Kanner et al, 1993;Blake et al, 1994;Somogyi et al, 1994), and production of IP3 (Sultan et al, 1991;Somogyi et al, 1994 (Hill et al, 1987;Ghosh et al, 1988;Harootunian et al, 1991).…”

“…Both bead adhesion and calcium signaling are specific to RGD and are decreased by antibodies that inhibit the function of av133 and avP35 integrins (Sjaastad et al, 1994 We examined the dependence of (Paulmichl et al, 1990). Ca2+ release occurs because of activation of phospholipase C and generation of IP3 via a phorbol ester and pertussis toxin-sensitive mechanism (Paulmichl et al, 1990 , 1993), tyrosine phosphorylation-dependent activation of phospholipase C (Kanner et al, 1993;Blake et al, 1994;Somogyi et al, 1994), and production of IP3 (Sultan et al, 1991;Somogyi et al, 1994 (Hill et al, 1987;Ghosh et al, 1988;Harootunian et al, 1991). MDCK cells were preloaded by electroporation in the presence of low-molecularweight heparin or unlabeled CS as a control (Boitano et al, 1992) (Figure 6c), whereas identical concentrations of CS had little or no effect (Figure 6d (Thastrup et al, 1990) Ca2" channels have been described (Fasolato et al, 1994) and that MDCK cells contain at least two known Ca2" entry pathways (Dietl and Volkl, 1994;Delles et al, 1995 (Figure 8b (Sjaastad et al, 1994 potent regulator of integrin-mediated adhesion to RGD than is Ca21 store release.…”

Purinergic signalling in the kidney in health and disease

“…Electrophysiological studies showed that ATP and UTP hyperpolarise MDCK cells by increasing the K + conductance [95,206]. A later paper from this group showed that ATP increased [Ca 2+ ] i ; calcium then activates K + channels and thus leads to hyperpolarisation of the cell membrane [281]. Regulation of transepithelial ion transport by two different receptors on the apical membrane of MDCK cells was claimed [430], the data implicating P2Y 1 and P2Y 2 (or P2Y 4 ) receptors.…”