Lorenzo Guerra

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na+/H+ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na+/H+ exchange of MDCK cell monolayers grown on permeant filter supports; pHi was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pHi (≈ 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na+/H+ exchange activity (Na+-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na+/H+ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na+/H+ exchange activity. An inhibitory effect of aldosterone was observed if Na+/H+ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na+ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na+/H+ exchange activity under conditions of low Na+, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na+ concentrations.

Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells

MDCK cells were grown to confluent monolayers on permeant filter supports; pH was analysed by using the pH-sensitive fluorescent probe 2′7′-biscarboxyethyl-5,6-carboxyfluorescein and a routine spectrofluorometer equipped with a perfusion cuvette [Krayer-Pawlowska et al. (1990) J Membr Biol 120:173–183]. Superfusion of the basolateral (but not apical) cell surface with Na+-containing solutions led to immediate recovery of pHi from an acid load (NH4 prepulse). This pHi recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent Km for Na+ of about 25 mM Na+ and an apparent Ki for inhibition by dimethylamiloride of around 0.2 μM; inhibition by dimethylamiloride was competitive with Na+ interaction. Lowering pHi prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent Vmax for Na/H exchange is increased more than tenfold by lowering the pHi from 7.0 to 6.7 without an effect on apparent Km values for Na+ interaction. It is concluded that MDCK cells (strain I) grown on a permeant support contain only basolateral Na/H exchange activity, most likely Na/H-1 [for nomenclature see Igarashi et al. (1991) Kidney Int 40:S84–S89].

Na+/H(+)-exchange in A6 cells: polarity and vasopressin regulation.

SummaryWe have analyzed the mechanism of Na+-dependent pHi; recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 2′7′-bis(2-carboxyethyl)5, 6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na+/H+-exchange activity only on the basolateral membrane: Na+/H+-exchange activity follows simple saturation kinetics with an apparent Kmfor Na+ of approximately 11 mm; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na+/H+-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na+/H+-exchange activity; AVP stimulates IP3 production at low concentrations, whereas much higher concentrations are required to stimualte cAMP formation. These findings suggest that in A6 cells (i) Na+/H+-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP3 turnover) is likely to be the mediator of AVP action at low AVP concentrations.

Vasopressin-dependent control of basolateral Na/H-exchange in highly differentiated A6-cell monolayers

We have used a well-differentiated A6-cell preparation (A6-C1) to study cellular location and vasopressin control of Na/H-exchange activity. After cell acidification, cell pHi (measured by BCECF-fluorescence) only recovered by the addition of Na medium to the basolateral cell surface; this pHi recovery was inhibited by dimethylamiloride (2 μm) consistent with basolateral location of Na/H-exchange activity. Addition of vasopressin produced stimulation of Na/H-exchange activity and increased the affinity of the exchanger for Na+. Stimulation of Na/H exchange was mimicked by pharmacological activation of protein kinase A (forskolin, 8-Br-cAMP) and not by pharmacological activation of protein kinase C (TPA). It is concluded that basolaterally located Na/H-exchange in A6-C1 cells is activated by vasopressin.