Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1)

Abstract

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.

Figure 1. Comparison of rates of Na⁺-H⁺ exchange activity in A6/NHE3 cells following repeated acidification of intracellular pH

Na⁺-H⁺ exchange activity was studied as Na⁺-dependent recovery of intracellular pH using microspectrofluorometry. For study of the influence of repeated acid loading on the rate of transport activity, confluent cell cultures were pulsed with NH₄Cl which was then replaced by TMA medium (onset of data presentation). Recovery of pH_i from an acid load was introduced by addition of Na⁺ medium. When rates of alkalinization approached original levels (data not shown), cells were again subjected to a protocol of NH₄Cl prepulse and withdrawal followed by Na⁺ addition. A, pH_i recovery in response to apical Na⁺ addition to acid-loaded cells; B, pH_i recovery in response to basolateral Na⁺ addition to acid-loaded cells. The tracings are representative of four similar experiments. TMA, perfusion with TMA medium; Na⁺, perfusion with Na⁺ medium.

Figure 2. Analysis of transcripts for NHE3 in A6/C1 and A6/NHE3 cells (clone 6 s and clone 9 s)

Total RNA was extracted from A6/C1 or A6/NHE3 cells and used to detect message for NHE3 (panel A) or AZUR4 (positive control; panel B) with RT-PCR. Southern blot hybridization (*, see below) to [³²P]-labelled, 3 kb NHE3 cDNA was then used to determine the identity of PCR products obtained with the NHE3 primer set (panel C). RT⁺, RT reactions performed in the presence of reverse transcriptase; RT⁻, RT reactions performed in the absence of reverse transcriptase. pCMV-5/NHE3_rat, PCR reaction performed with the linearized expression plasmid containing the full sequence of rat NHE3; M, molecular mass marker (100 bp ladder from Life Technologies, Gibco). *Note that the band in lane 8 of the Southern blot represents hybridization of pCMV-5/NHE3 to 3 kb NHE3 cDNA, because PCR probes were run on a single agarose gel with an upper and lower slit row.

Figure 3. Na⁺ dependence and polarity of pH_i recovery in acid-loaded A6/C1 and A6/NHE3 cells

Confluent cell cultures of A6/C1 or A6/NHE3 cells on filter support were examined for pH_i change by microspectrofluorometry using the dye BCECF as detailed in Methods. Cells in Na⁺ medium were pulsed with 40 mM NH₄Cl and then perfused with Na⁺-free medium (TMA). Recovery of pH_i from an acid load was next induced by re-introduction of Na⁺ medium (Na⁺) to either the apical or the basolateral cell surface. Fluorescence ratio traces from A6/C1 cells (A) and from A6/NHE3 cells (B), representative of at least four independent experiments.

Figure 4. Modulation of NHE activity by a PKA agonist/antagonist

Na⁺-H⁺ exchange activity was assessed as initial rates of HOE694 inhibitable pH_i recovery of A6 cell cultures from an acid load as described in Fig. 3. To test the effect of 8-bromo-cAMP (PKA agonist) and/or H89 (PKA antagonist), cell cultures were repetitively acidified to the same starting acid pH_i value, and pH_i recovery rates were observed either in the absence or presence of pharmacological agents added to the apical and basolateral cell surface. In all experiments, cells were exposed to different agents for 15 min prior to evaluating their effect on Na⁺-H⁺ exchange activity in the absence or presence of HOE694 that was included for a 2 min period in the apical perfusate. The number of experiments performed under identical experimental conditions is given in parentheses. ap, the change (expressed in %) of the activity of apical Na⁺-H⁺ exchanger in response to pharmacological agents; bl, the corresponding change (expressed in %) of basolateral Na⁺-H⁺ exchanger; HOE694 ‘resistant’, agonist-induced net change (expressed in %) of apical transport activity which was calculated as difference between the sum of Na⁺-dependent rate of pH_i recovery measured in the presence of agonists and 10⁻⁴ M apical HOE694 and 10⁻⁴ M apical HOE694 alone. * Significant vs. control;⁺ significant vs. HOE694;. significant vs. 8-bromo-cAMP; ▴ significant vs. 8-bromo-cAMP plus HOE694.

Figure 5. Modulation of NHE activities by a PKC agonist/antagonist

The effect of PMA (PKC agonist) and/or calphostin C (PKC antagonist) on Na⁺-H⁺ exchange activities of confluent A6/NHE3 cells was tested as indicated in the legend to Fig. 4. Exposure to different agents (which were included in the apical and basolateral perfusate) was for 15 min, except for HOE694 which was added to the apical perfusate 2 min prior to evaluating the influence of different agents on Na⁺-dependent rate of alkalinization. The number of experiments performed under identical experimental conditions is given in parentheses. ap, change (expressed in %) of the activity of apical Na⁺-H⁺ exchanger in response to pharmacological agents; bl, corresponding change (expressed in %) of basolateral Na⁺-H⁺ exchanger; HOE694 ‘resistant’, change (expressed in %) of apical transport rates in response to pharmacological agents and HOE694 when normalized to inhibition of 10⁻⁴ M HOE694 alone. * Significant vs. control; ⁺ significant vs. HOE694;. significant vs. PMA; ▴ significant vs. PMA plus HOE694.

Figure 6. CPA effects on NHE activities of A6/NHE3 cells in clone 6 s, in the absence and presence of PKC or PKA antagonists

A, effect of apical CPA (adenosine analogue) on rate of Na⁺-H⁺ exchanger. B, effect of basolateral CPA on rate of Na⁺-H⁺ exchanger. NHE activities were observed under control and test conditions as indicated in Fig. 4. Except for CPA, agents were applied to the apical and basolateral aspect of the cells. Changes in rate of NHE activities were observed 15 min after application of pharmacological agents in the presence or absence of HOE694 (which was added to the apical perfusate 2 min prior to initiation of Na⁺-dependent cell alkalinization). The number of experiments performed under identical experimental conditions is given in parentheses. ap, change (expressed in %) of the activity of apical Na⁺-H⁺ exchanger in response to pharmacological agents; bl, corresponding change (expressed in %) of basolateral Na⁺-H⁺ exchanger; HOE694 ‘resistant’, change (expressed in %) of apical transport rates in response to pharmacological agents and HOE694 when normalized to inhibition of 10⁻⁴ M HOE694 alone. * Significant vs. control;⁺ significant vs. HOE694;. significant vs. CPA; ▴ significant vs. CPA plus HOE694.