Parathyroid hormone regulation of Na+/H+ exchange in opossum kidney cells: polarity and mechanisms

In previous work we have shown that parathyroid hormone (PTH) inhibits Na⁺/H⁺ exchange in cellular suspensions of OK (oppossum kidney) cells (an established renal epithelial cell line) in a dose-dependent manner. PTH effects could be mimicked by pharmacological activation of both protein kinase A and protein kinase C (Helmle-Kolb et al. 1990). In the present paper we extend these observations and analyze the PTH-dependent control of Na⁺/H⁺ exchange in OK cells kept in epithelial configuration (monolayer). Na⁺/H⁺ exchange activity is examined by microfluorometry using the intracellularly trapped pH-sensitive dye 27-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. Cells recovered from an acid load (NH₄Cl prepulse) after addition of apical Na⁺. Ethylisopropylamiloride inhibits Na⁺-dependent pH_i recovery at micromolar concentrations. PTH leads to an inhibition of apical Na⁺/H⁺ exchange activity; inhibition is observed even at a concentration of 5 pM PTH. PTH given at maximally effective concentrations (24 nM) reduces the total Na⁺/H⁺ exchange capacity by 60%–70%. Apical as well as basolateral hormone additions elicit an inhibitory response at low (5 pM) or high (24 nM) concentrations. Forskolin (activation of protein kinase A) and phorbol esters (activation of protein kinase C) lead to an inhibition of Na⁺/H⁺ exchange activity (60%–70% inhibition). These observations suggest that Na⁺/H⁺ exchange activity is preferentially located in the apical membranes of OK cells kept in monolayer configuration. Na⁺/H⁺ exchange activity is partially inhibited by PTH (even at physiological hormone concentrations) and PTH-dependent inhibition can be mimicked by pharmacological activation of either protein kinase A or protein kinase C. Interestingly, PTH-related regulatory mechanisms are induced from both the apical and basolateral cell surface suggesting a location of receptors at both cell surfaces.     

Polarized expression of Na+/H+ exchange activities in clonal LLC-PK1 cells (Clone4 and PKE20)

We have analysed the mechanisms of Na⁺-dependent pH_i recovery from an acid load in LLC-PK₁/ Clone₄ and LLC-PK₁/PKE₂₀ cells by using the intracellular pH indicator 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester. By analysis using single-cell microspectrofluorometry, we obtained evidence for polarized expression of Na⁺/H⁺ exchange activities with different properties in apical and basolateral cell surfaces, respectively. In Clone₄ cells, Na⁺/H⁺ exchange activity is only visible on the basolateral cell surface; in PKE₂₀ cells, Na⁺/H⁺ exchange activities with equal capacities are present on both cell surfaces. In Clone₄ cells, the apparent K _m value for Na⁺ is around 10 mM; in PKE₂₀ cells it is around 20 mM and indistinguishable for the two cell poles. Ethylisopropylamiloride (EIPA) inhibition for all three activities measured in monolayer configuration is reduced by increasing Na⁺ concentration. Measured in the same cells, EIPA inhibition of transport of PKE₂₀ cells is weaker for apical Na⁺/H⁺ exchange as compared to basolateral activity. In Clone₄ and PKE₂₀ cells kept in suspension, Na⁺/H⁺ exchange activities with similar properties for the two cell lines are observed. However, Na⁺/H⁺ exchange activities in cells in suspension are different from either activity measured in monolayer configuration: affinity for Na⁺ is higher (PKE₂₀ cells) and inhibition by amiloride is weak and not influenced by increasing Na⁺ concentrations (PKE₂₀ and Clone₄ cells). It is concluded that PKE₂₀ cells contain different Na⁺/H⁺ exchange activities on the two cell surfaces; this cell line should be a useful model to study regulatory aspect of different Na⁺/H⁺ exchange functions (epithelial/housekeeping). Furthermore, measurements on cells in suspension do not reflect the properties of Na⁺/H⁺ exchange activities of cells in epithelial configuration.     

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 27-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 pH_i from an acid load (NH₄ prepulse). This pH_i recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent K _m for Na⁺ of about 25 mM Na⁺ and an apparent K _i for inhibition by dimethylamiloride of around 0.2 M; inhibition by dimethylamiloride was competitive with Na⁺ interaction. Lowering pH_i prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent V _max for Na/H exchange is increased more than tenfold by lowering the pH_i from 7.0 to 6.7 without an effect on apparent K _m 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].     

Activity and expression of Na+/H+ exchanger isoforms in the syncytiotrophoblast of the human placenta

The purpose of this study was to compare Na⁺/H⁺ exchanger (NHE) activity in the microvillous (MVM) and basal (BM) plasma membrane of the human placental syncytiotrophoblast and to determine the relative contribution of various NHE isoforms to this activity. Uptake of ²²Na into isolated MVM vesicles in the presence of a H⁺ gradient, at initial rate, was four- to fivefold higher than that by BM vesicles (214±28 vs. 49±9 pmol/mg protein per 30 s, respectively, means±SEM, n=8, 6, P<0.001). The ²²Na uptake by MVM, but not by BM, was reduced in the absence of a H⁺ gradient and in the presence of 500 M amiloride. To determine the contribution of NHE1, NHE2 and NHE3 isoforms to NHE activity in MVM, we investigated the effect of amiloride analogues which show isoform selectivity. HOE 694, an analogue selective for NHE1 at low concentrations, inhibited ²²Na uptake with an EC₅₀ of 0.13±0.05 M (n=6), whereas S3226, an analogue selective for NHE3 at low concentrations had an EC₅₀ of 3.01±0.85 M (n=5). To investigate this further, we measured recovery of syncytiotrophoblast intracellular pH (pH_i) from an acid load using a H⁺-selective, fluorescent dye (BCECF) loaded into isolated intact placental fragments. This recovery was blocked in the absence of Na⁺ and the presence of amiloride (500 M) and concentrations of HOE 694 and S3226 were comparable to those used in vesicle experiments. Overall these data show that under the conditions used NHE activity in the term placental syncytiotrophoblast is absent from BM. NHE activity in the MVM is attributable predominantly to NHE1.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Stimulation of Na+/H+ exchange activity by endothelin in opossum kidney cells

Endothelin-1 (ET-1) controls multiple aspects of kidney function. In this study we have analysed the effects of ET-1 on apical Na⁺/H⁺ exchange activity in opossum kidney (OK) cells. ET-1 (at 10^–10 M and 10^–8 M) activated Na⁺/H⁺ exchange activity within 5 min of exposure. ET-1 (10^–8 M) prevented PTH-induced (parathyroid hormone; 10^–8 M) inhibition of Na⁺/H⁺ exchange activity; it also abolished transport inhibition in response to 10^–3 M IBMX (isobutylmethylxanthine) and 3×10^–7 M TPA (phorbol 12-myristate 13-acetate), but had no effect on the 8-bromo-cAMP-induced (10^–4 M) decrease of transport rate. Basal cAMP content, IBMX- and PTH-stimulated cAMP production were unaffected by ET-1 (10^–8 M). The stimulatory action of ET-1 (10^–8 M) on Na⁺/H⁺ exchange activity was prevented by calphostin C (10^–8 M). These data document that OK cells might serve as a useful in vitro model for analysis of cellular mechanisms involved in endothelin action; proteine kinase C activation seems to participate in the observed endothelin effects.     

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid     

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

Studies with apical membrane vesicles have shown that two distinct and separate anion exchange processes are present in rat distal colon, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive exchange, and DIDS-resistant Cl⁻–OH⁻ exchange. These studies proposed that anion exchanger (AE)-1 isoform encodes the former as both apical membrane DIDS-sensitive exchange, and AE1 specific mRNA are present only in surface cells and are downregulated in Na-depleted rats, whereas downregulated in adenoma (DRA) encodes the latter as both DIDS-resistant Cl⁻–OH⁻ exchange, and DRA-specific proteins are present in apical membranes of both surface and crypt cells and are not altered in Na⁺-depleted rats. Studies were, therefore, initiated to identify the function of rat DRA (rDRA) in vitro. rDRA cDNA isolated from rat distal colon encodes a 757-amino-acid protein which has 96 and 81% homology with mDRA and hDRA, respectively. rDRA-specific mRNA expression was detectable only in specific segments of the digestive tract (duodenum, ileum, cecum, proximal colon, and distal colon) but not in the stomach, jejunum, or in the kidney, brain, heart, and lung. HEK 293 cells stably transfected with rDRA exhibited DIDS-insensitive and intracellular acid pH (pH_i 6.5)-sensitive Cl uptake that: (1) was significantly stimulated by outward Cl⁻, , isobutyrate, and possibly OH⁻ gradients; (2) was saturated as a function of increasing extracellular Cl concentrations with an apparent K _m for Cl of 2.9 ± 0.3 mM; and (3) was inhibited competitively by extracellular oxalate but not by . A high rate of DIDS-insensitive Cl influx at pH 6.5 was also present under physiological Cl⁻ concentration. Our observations that rDRA mediates DIDS-insensitive, acid pH-dependent Cl⁻ uptake are consistent with prior observations that rDRA does not mediate DIDS-sensitive exchange in rat distal colon. We speculate that, in addition to mediating pH-sensitive Cl⁻ uptake, rDRA may function as a modifier of other anion transport proteins.     

Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

The mechanism of regulation of intracellular pH (pH_i) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2,7-bis(2-carboxyethyl)-5(and –6)-carboxyfluorescein. The pH_i in the TRIS/HEPES-buffered standard solution was 7.29±0.01. Addition of 1 mol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pH_i. These agents decreased pH_i in the absence of external Na⁺ or in the presence of amiloride. The rate of pH_i recovery from an acid load after NH ₄ ⁺ prepulse was a linear function of pH_i and increased as pH_i became more acidic. Addition of ACh shifted the relationship towards a more alkaline pH_i range. The increase in pH_i induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mol/l). Addition of 0.1–1 mol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pH_i within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pH_i. In Ca²⁺-free solution with 50 mol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pH_i and cytosolic Ca²⁺ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na⁺/H⁺ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na⁺/H⁺ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na⁺/H⁺ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca²⁺ concentration. The implication of the cytosolic acidification and cell volume change in pH_i regulation is discussed.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Gene expression of the human prostaglandin E receptor EP4 subtype: differential regulation in monocytoid and lymphoid lineage cells by phorbol ester

We isolated a cDNA clone encoding the human prostaglandin (PG) E receptor EP₄ subtype and examined the gene expression in human blood cells. Northern blot analysis revealed that the EP₄ gene is expressed at a high level in peripheral blood mononuclear cells, and at lower levels in cultured human blood cell lines, THP-1 and U937 (monocytoid cell lines), MOLT-4 and Jurkat (T-cell lines), and Raji (B-cell line). To examine regulation of the EP₄ gene expression in the immune system, we studied the effects of phorbol 12-myristate 13-acetate (PMA) on these cell lines. Gene expression was upregulated in THP-1, U937, and Raji cells by PMA, and was downregulated in MOLT-4 and Jurkat cells. In THP-1 cells the effects of PMA were further analyzed, and the upregulation of the EP₄ gene was shown to be followed by an increase in PGE₂ binding sites and in PGE₂-induced cAMP accumulation. In the striking contrast, other PGE receptor subtypes (EP₁, EP₂ and EP₃) and other prostanoid receptors (IP and DP) were shown not to be upregulated by PMA. Therefore, this is the first demonstration of a highly specific upregulation of the EP₄ subtype in THP-1 cells treated with PMA, suggesting the importance of the EP₄ subtype in the immune system. In the present study we also clarified that EP₄ gene expression is regulated differently among human monocytoid and lymphoid lineage cells, thus leading to the better understanding of the regulatory mechanisms for the human EP₄ gene expression in the immune system.Abbreviations PG Prostaglandin - PMA Phorbol 12-myristate 13-acetate - PBMC Peripheral blood mononuclear cells     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.