Corinna Helmle-Kolb

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na+/phosphate co-transport and Na+/H+ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na+/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na+/H+ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca2+ concentration do not induce inhibition of Na+/H+ exchange; PTH-dependent inhibition of Na+/H+ exchange is not prevented by ionomycin or by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Ca2+ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40–50% inhibition of Na+/H+ exchange. An analysis of intracellular pH sensitivity of Na+/H+ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced Vmax of Na+/H+ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na+/H+ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).

Effect of short-chain fatty acids on cell volume and intracellular pH in rat distal colon.

Superfusion of isolated crypts from the rat colon with sodium-butyrate-containing solutions induced an increase in the crypt diameter indicating a swelling of the crypt cells. The response to butyrate (50 mmol 1−1) was not uniform along the crypt axis, the most pronounced swelling being observed in the upper third of the crypt. The butyrate effect was concentration-dependent and was completely suppressed by amiloride, suggesting that it is caused by activation of the Na+/H+ exchanger. Acetate, propionate and isobutyrate had a similar action. In HEPES-buffered solution the butyrate-induced change in cell volume was monophasic, i. e. only a swelling took place, whereas in HCO3− buffer it was biphasic, i. e. swelling was followed by a regulatory volume decrease. This decrease was suppressed by K+ and Cl− channel blockers as well as inhibitors of leukotriene synthesis. Measurements of intracellular pH with the fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) revealed that butyrate induced an acidification of the cell, which was stronger in HEPES than in HCO3− buffer. Estimation of Na+/H+ exchange activity, tested as recovery of intracellular pH from an acid load via an NE4Cl prepulse, revealed a much lower Na+/H+ exchange activity in the fundus region compared to the upper third of the crypt. The smaller volume response evoked by butyrate in the fundus region probably reflects the smaller Na+/H+ activity compared to the more differentiated cells near the opening of the crypt. It is concluded that cell swelling caused by short-chain fatty acids is a physiological stimulus for volume regulation. This response is restricted to the more differentiated cells.

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors Is Mediated by Calcineurin Homologous Protein

Adenosine is an autacoid that regulates renal Na+ transport. Activation of adenosine A1 receptor (A1R) by N6-cyclopentidyladenosine (CPA) inhibits the Na+/H+ exchanger 3 (NHE3) via phospholipase C/Ca2+/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A1R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10-6 M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3C terminus; prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3C terminus (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3Δ552) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3462-552) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3Full-length interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A1R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.

Bimodal Acute Effects of A1 Adenosine Receptor Activation on Na+/H+ Exchanger 3 in Opossum Kidney Cells

Regulation of renal apical Na+/H+ exchanger 3 (NHE3) activity by adenosine has been suggested to contribute to acute control of mammalian Na(+) homeostasis. The mechanism by which adenosine controls NHE3 activity in a renal cell line was examined. The adenosine analog, N(6)-cyclopentyladenosine (CPA) exerts a bimodal effect on NHE3: CPA concentrations >10(-8) M inactivate NHE3, whereas concentrations <10(-8) M stimulate NHE3 activity. Acute CPA-induced control of NHE3 was blocked by antagonists of A1 adenosine receptors and inhibition of phospholipase C, pretreatment with BAPTA-AM (chelator of cellular calcium), and exposure to pertussis toxin. Stimulatory and to some extent also inhibitory CPA concentrations attenuated 8-bromo-cAMP and dopamine-mediated inhibition of NHE3. BAPTA eliminated the ability of a stimulatory dose of CPA to attenuate 8-bromo-cAMP-induced suppression of NHE3 activity. Upon inhibition of protein kinase C, CPA at an inhibitory dose provoked activation of NHE3, which is partially reverted by 8-bromo-cAMP and suppressed by pre-incubation with BAPTA-AM. Cytochalasin B, an actin-modifying agent, selectively prevented downregulation but did not affect upregulation of NHE3 activity by CPA. In conclusion, these observations demonstrate that (1) CPA modulates NHE3 activity by elevation of cellular Ca(2+) exerting a negative control on adenylate cyclase activity, (2) protein kinase C is the determining factor leading to CPA-induced downregulation of NHE3 activity, and (3) alterations of surface NHE3 abundance may contribute to A1 adenosine receptor-dependent inhibition of NHE3 activity.

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.

Studies on the kinetics of Na+/H+ exchange in OK cells: Introduction of a new device for the analysis of polarized transport in cultured epithelia

SummaryThe present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na+/H+ exchange at the level of an intact monolayer. Na+/ H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+/H+ exchange activity was measured by monitoring changes in intracellular pH (pHi) after an acid load, using the pH-sensitive dye 2′7′-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pHi, recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pHi=7.68; pHo=7.4) where Na+/H+ exchange is not detectable, transport rate increased as pHi decreased. A positive cooperativity characterized the interaction of internal H+ with the exchanger, and suggests multiple H+ binding sites. In contrast, extracellular [Na+] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na+ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and theKi was 3.4 μM. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pHi and processes associated with their polarized expression and regulation.

Na/H exchange activities in NHE1-transfected OK-cells : cell polarity and regulation

The human fibroblast, “amiloride-sensitive” Na/H exchanger (NHE1) was transfected into opossum kidney cells (OK cells) (OK/NHE1 cells). Northern blot analysis confirmed that the NHE1 message is expressed in OK/NHE1 cells. In immunoblot analysis, an anti-human NHE1 antibody labelled a membrane protein only present in OK/NHE1 cells. In contrast to the parental cell line containing only an apically located, “amilorideresistant” Na/H exchange activity, OK/NHE1 cells contain apically and basolaterally located Na/H exchange activities, the apical activity being “amiloride resistant” and the basolateral being “amiloride sensitive”. Parathyroid hormone (PTH) inhibited apical transport activity (OK and OK/NHE1 cells) but had no effect on basolateral transport activity (OK/NHE1 cells). Pharmacological activation of protein kinase A (forskolin) decreased both apical and basolateral Na/H exchange activity. Incubation with phorbol ester (exogenous activation of protein kinase C) reduced apical Na/H exchange activity (OK and OK/NHE1 cells) but had only a moderate, inhibitory effect on basolateral Na/H exchange activity (OK/NHE1 cells). These results indicate that transfection of OK cells with human fibroblast NHE1 cDNA encoding an “amiloride-sensitive” form of the Na/H exchanger results in expression of basolaterally located “NHE1-related” transport activity. Regulatory control of intracellular Na/H exchange activities (apically versus basolaterally located) and intercellular Na/H exchange activities (NHE1-related) differs. This may relate to cell-specific properties as well as to exchanger-specific properties.

Identification of PTH-responsive Na/H-exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2)

Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2′,7′,bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pHi) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pHi recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/ H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10−8 M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3′5′-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities. Examination of the intracellular Ca2+ concentration ([Ca2+]i; using fura-2) and cAMP content (using a cAMP binding protein assay) revealed only PTH-dependent stimulation of adenylate cyclase. These results suggest that PTH (mediated by protein kinase A) inhibits in RKPC-2 cells structurally distinct Na/H-exchange activities (NHE-2 and NHE-1); we assume apical location of NHE-2 (‘amiloride-resistant’) and basolateral location of NHE-1 (‘amiloride-sensitive’) isoforms of Na/H-exchange activities.

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH)

SummaryKidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10−8m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10−8m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10−8m) resulted in a rapid and transient increase in [Ca2+]i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca2+ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/Pi cotransport.

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

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 N6‐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.