Biomedical subjects

J Orlowski

Publications and source records attributed to J Orlowski.

At least 19 recordsLinked to original sources

PubMed1996

Galpha12 differentially regulates Na+-H+ exchanger isoforms.

Activation of several GTPases stimulates Na+-H+ exchange, resulting in an increased efflux of intracellular H+. These GTPases include alpha subunits of the heterotrimeric G proteins Gq and G13, as well as the low molecular weight GTP-binding proteins Ras, Cdc42, and Rho (Hooley, R., Yu, C.-Y., Simon, M., and Barber, D. L. (1996) J. Biol. Chem. 271, 6152-6158). GTPases coupled to the inhibition of Na+-H+ exchange, however, have not been identified. Several neurotransmitters, including somatostatin and dopamine, inhibit Na+-H+ exchange through a guanine-nucleotide-dependent mechanism, suggesting the involvement of a GTPase. In this study we determined that mutational activation of the alpha subunit of G12 inhibits the ubiquitously expressed Na+-H+ exchanger isoform, NHE1. Transient expression of mutationally activated Galpha12 inhibited serum- and Galpha13-stimulated NHE1 activity in HEK293 cells and CCL39 fibroblasts. In addition, in NHE-deficient AP1 cells stably expressing specific NHE isoforms, mutationally activated Galpha12 inhibited NHE1 activity but stimulated activities of the Na+-H+ exchanger (NHE) isoforms NHE2 and NHE3. In contrast, mutationally activated Galpha13, another member of the Galpha12/13 family, stimulated all three NHE isoforms. Although previous studies have identified a parallel action of Galpha12 and Galpha13 in regulating MAP (mitogen-activated protein) kinases and cell growth, these GTPases have opposing effects on NHE1 activity.

Animals↗

PubMed1996

Delineation of transmembrane domains of the Na+/H+ exchanger that confer sensitivity to pharmacological antagonists.

Plasma membrane Na+/H+ exchanger (NHE) isoforms NHE1 and NHE3 exhibit very different sensitivities to amiloride and its 5-amino-substituted analogues, benzoyl guanidinium derivatives (e.g. (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE694)), and cimetidine. To define structural domains that confer differential sensitivity to these antagonists, unique restriction endonuclease sites were engineered into cDNAs for each isoform near the regions that encode the putative membrane-spanning domains. These new sites did not modify their pharmacological properties and allowed several chimeric Na+/H+ exchangers to be constructed by exchanging homologous segments. The modified parental (E1' and E3') and chimeric molecules were stably expressed in exchanger-deficient Chinese hamster ovary AP-1 cells and assayed for their sensitivities to amiloride, ethylisopropylamiloride, HOE694, and cimetidine. Most chimeras showed drug sensitivities corresponding to the dominant parental segment. However, interchanging a 66-amino acid segment containing the putative ninth transmembrane (M9) domain and its adjacent loops caused reciprocal alterations in the sensitivities of E1' and E3' to all antagonists. In addition, substituting the first five putative membrane-spanning domains of E3' with the corresponding region of E1' modestly reduced the transporter's sensitivity to cimetidine but not the other compounds. These data indicate that the protein segment between M8 and M10 may be a major site of interaction with these antagonists, although other regions modestly influence sensitivity to certain drugs.

Amiloride↗

PubMed1996

Structurally diverse N-terminal peptides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) inhibit the Na+/H+ exchanger NHE3 isoform by binding to the PTH/PTHRP receptor type I and activating distinct signaling pathways.

N-terminal peptides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) elicit a wide variety of biological responses in target cells, including the inhibition of Na+/H+ exchanger NHE3 activity in renal cells. This response is believed to be mediated by ligand binding to a common receptor (i.e. PTH/PTHRP receptor type I) and activation of cAMP-dependent and/or Ca2+/phospholipid-dependent protein kinases (PKA and PKC, respectively). However, the mechanism of action of these N-terminal peptides is now unclear because of recent data reporting the existence of additional receptor isoforms. Therefore, to directly examine the ligand binding and signaling characteristics of the PTH/PTHRP receptor type I and its ability to elicit a biological response, cDNAs encoding the rat type I receptor and the rat NHE3 isoform were transfected into Chinese hamster ovary (AP-1) cells that lack endogenous expression of these proteins. Competition binding assays using [125I-Tyr36]PTHRP-(1-36)-NH2 radioligand indicated that several biologically active human N-terminal PTH and PTHRP fragments (PTH-(1-34), PTH-(3-34), PTH-(28-42), PTH-(28-48), and PTHRP-(1-34)) were capable of binding to the type I receptor. Both PTH-(1-34) and PTHRP-(1-34) stimulated adenylate cyclase and PKC activities in these cells, whereas PTH-(3-34), PTH-(28-42), and PTH-(28-48) selectively enhanced only PKC activity. PTHRP-(1-16), a biologically inert fragment, was incapable of binding to this receptor and influencing either the PKA or PKC pathway. Furthermore, all the analogues with the exception of PTHRP-(1-16) inhibited NHE3 activity. Inhibition of PKC by the potent antagonist chelerythrine chloride abolished the depression of NHE3 activity by PTH-(3-34), PTH-(28-42), and PTH-(28-48) but did not alleviate the effects of PTH-(1-34). Likewise, antagonism of PKA by H-89 was unable to prevent the inhibition caused by PTH-(1-34). However, inhibition of both PKA and PKC by the nonselective protein kinase antagonist H-7 abolished the reduction of NHE3 activity by PTH-(1-34). These data indicate that discrete N-terminal analogues of PTH and PTHRP can interact with the classical PTH/PTHRP receptor type I and activate PKA and/or PKC. Activation of either signaling pathway independently leads to inhibition of NHE3.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Genomic organization and glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene.

The activity of the apical membrane Na+/H+ exchanger NHE3 isoform of renal or intestinal epithelial cells is chronically regulated by a wide variety of stimuli, including acidosis, cAMP, glucocorticoids, and thyroid hormone. To understand the molecular mechanisms responsible for long term regulation of this cation transporter, we have isolated and determined the structure of this gene from a rat genomic library. The Nh3 gene spans > 40 kilobases and contains 17 exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The transcription initiation site was mapped by S1 nuclease protection analyses of mRNA from rat kidney and intestine. Multiple start sites were clustered between nucleotides -100 and -96 relative to the translation initiation codon. An atypical TATA-box and CCAAT-box are centered 30 and 147 nucleotides, respectively, upstream of the predominant transcription initiation site. Sequence analysis of approximately 1.4 kilobases of the 5'-flanking promoter region also revealed the presence of other putative cis-acting elements recognized by various transcription factors (e.g. AP-1, AP-2, C/EBP, NF-I, OCT-1/OTF-1, PEA3, Sp1, glucocorticoid, and thyroid hormone receptors), some of which may participate in the chronic regulation of this gene. The glucocorticoid responsiveness of the Nhe3 gene was assessed by fusing its 5' regulatory region to the firefly luciferase reporter gene and then by measuring the expression of the chimeric gene in transiently transfected renal epithelial OK and LLC-PK1 cells. Glucocorticoid treatment significantly increased the luciferase activity of the chimeric gene in both cell lines, thereby indicating that glucocorticoid regulation of Nhe3 is mediated primarily by a transcriptional mechanism.

Amino Acid Sequence↗

PubMed1996

Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform.

Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N-terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.

Adenosine Triphosphate↗

PubMed1996

Transient expression of Na+/H+ exchanger isoform NHE-2 in LLC-PK1 cells: inhibition of endogenous NHE-3 and regulation by hypertonicity.

Na+/H+ exchanger isoforms NHE-2 and NHE-3 demonstrate distinct tissue expression patterns in renal epithelial cells. NHE-2 is predominantly expressed in the inner medulla whereas NHE-3 is highly expressed in the proximal tubule cells. The purpose of the current experiments was to study the characteristics of NHE-2 upon its own expression in cultured proximal tubule cells, LLC-PK1. Toward this end, LLC-PK1 cells were subjected to six cycles of proton suicide. The mutant cells, when grown to confluence and assayed for Na+/H+ exchanger by 22Na+ influx, showed significant reduction in NHE activity as compared to the parent cells (10.4 nmole/mg prot/4 min in parent cells vs. 1.8 in mutant cells, P < 0.001, n = 4). This remaining exchanger activity was mostly mediated via NHE-3 as shown by inhibition of the Na influx following PKC stimulation (65% with PMA vs. 100% without PMA. P < 0.05, n = 4). The mutant cells were transiently transfected with a pCMV/NHE-2 expression vector using calcium phosphate precipitation method. Northern blot analysis showed the expression of a 3.4 kb transcript only in the transfected cells. The expression peaked at 48 hr and diminished by 96 hr. The exchanger activity at 48 hr after transfection was mostly due to NHE-3 (as shown by inhibition in the presence of PMA) but was significantly lower than in sham transfected cells (1.2 nmoles/mg prot. in NHE-2-transfected and 2.1 in sham-transfected, P < 0.05, n = 4). At 60 hr after transfection, the cells exhibited PMA-stimulated Na influx (>28%) indicating functional expression of NHE-2. Increasing the osmolality of the media to 510 mOsm/l stimulated the Na+/H+ exchanger in NHE-2 transfected cells but inhibited the exchanger activity in sham transfected cells. In conclusion, NHE-2 appears as a 3.4 kb transcript in transfected LLC-PK1 cells and functional expression of NHE-2 is preceded by inhibition of endogenous NHE-3 activity. The NHE-2 is stimulated by hypertonicity, indicating a likely role for this isoform in cell volume regulation.

Animals↗

PubMed1996

Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases.

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.

Amiloride↗

PubMed1995

Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.

Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.

Animals↗

PubMed1995

Cytoplasmic domain of the ubiquitous Na+/H+ exchanger NHE1 can confer Ca2+ responsiveness to the apical isoform NHE3.

The Na+/H+ exchanger isoforms NHE1 and NHE3 are regulated differently by various stimuli. Calcium has been recognized as one of the major second messengers in such exchanger regulation. We previously proposed that Ca(2+)-induced activation of NHE1 occurs via displacement of its autoinhibitory domain from the H+ modifier site due to direct binding of Ca2+/calmodulin. To further validate this hypothesis, the functional role of the cytoplasmic domain was studied in both wild-type and chimeric exchangers, i.e. NHE1, NHE3, NHE1 with the cytoplasmic domain of NHE3 (N1N3), and NHE3 with the cytoplasmic domain of NHE1 (N3N1). After expression in exchanger-deficient fibroblasts (PS120), early response (< 80 s) to external stimuli was assessed as 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Among stimuli tested (ionomycin, alpha-thrombin, phorbol ester, hyperosmotic stress, and platelet-derived growth factor) that are all known to activate NHE1, only ionomycin and thrombin induced a significant intracellular Ca2+ mobilization and early activation of 22Na+ uptake, implying that Ca2+ is a main regulator of NHE1 in the early phase of the agonist response. However, all the stimuli did not activate NHE3 or N1N3. In contrast, a significant stimulation of 22Na+ uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pHi sensitivity (approximately 0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pHi sensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca(2+)-induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H(+)-modifier site of a different isoform, NHE3.

Amiloride↗

PubMed1995

Parathyroid hormone and parathyroid hormone-related peptide activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic cells (UMR-106) via a cAMP-dependent pathway.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na+/H+ exchanger activity in osteoblastic cells, although the signaling components involved are not precisely defined. Since these peptide hormones can stimulate production of diverse second messengers (i.e. cAMP and diacylglycerol) that activate protein kinase A (PKA) and protein kinase C (PKC) in target cells, it is conceivable that either one or both of these pathways can participate in modulating exchanger activity. To discriminate among these possibilities, a series of synthetic PTH and PTHRP fragments were used that stimulate adenylate cyclase and/or PKC. In the osteoblastic cell line UMR-106, human PTH(1-34) and PTHRP(1-34) augmented adenylate cyclase activity, whereas PTH(3-34), PTH(28-42), and PTH(28-48) had no effect. Nevertheless, all these peptide fragments were found to enhance PKC translocation from the cytosol to the membrane in a dose-dependent (10(-11) to 10(-7) M) manner. PTHRP(1-16), a biologically inert fragment, was incapable of influencing either the PKA or PKC pathway. PTH(1-34) and PTHRP(1-34), but not PTH(3-34), PTH(28-42), PTH(28-48), or PTHRP(1-16), elevated Na+/H+ exchanger activity, implicating cAMP as the transducing signal. In accordance with this observation, forskolin (10 microM), which directly stimulates adenylate cyclase, also activated Na+/H+ exchanger activity. The involvement of PKA was verified when the highly specific PKA inhibitor, H-89, completely abolished the stimulatory effect of PTH(1-34) and forskolin on Na+/H+ exchange. In addition, Northern blot analysis revealed the presence of only the NHE-1 isoform of the Na+/H+ exchanger in UMR-106 cells. In summary, these results indicated that PTH and PTHRP activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic UMR-106 cells exclusively via a cAMP-dependent pathway.

Animals↗

PubMed1995

Parathyroid hormone and parathyroid hormone-related peptide inhibit the apical Na+/H+ exchanger NHE-3 isoform in renal cells (OK) via a dual signaling cascade involving protein kinase A and C.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) interact with a common G protein-coupled receptor and stimulate production of diverse second messengers (i.e. cAMP, diacylglycerol, and inositol 1,4,5-trisphosphate) that varies depending on the target cell. In renal proximal tubule OK cells, PTH inhibits the activity of the apical membrane Na+/H+ exchanger, although it is unclear whether the signal is transmitted through protein kinase A (PKA) and/or protein kinase C (PKC). To delineate the signaling circuitry, a series of synthetic PTH and PTHRP fragments were used that stimulate the adenylate cyclase-cAMP-PKA and/or phospholipase C-diacylglycerol-PKC pathways. Human PTH-(1-34) and PTHRP-(1-34) stimulated adenylate cyclase and PKC activity, whereas the PTH analogues, PTH-(3-34), PTH-(28-42), and PTH-(28-48), selectively enhanced only PKC activity. However, each peptide fragment inhibited Na+/H+ exchanger activity by 40-50%, suggesting that PKC and possibly PKA were capable of transducing the PTH/PTHRP signal to the transporter. This was corroborated when forskolin and phorbol 12-myristate 13-acetate (PMA), direct agonists of adenylate cyclase and PKC, respectively, both inhibited the Na+/H+ exchanger. The specific PKA antagonist, H-89, abolished the forskolin-mediated suppression of Na+/H+ exchanger activity, but did not prevent the inhibitory effects of PTH-(1-34) or PMA. In comparison, the potent PKC inhibitor, chelerythrine chloride, prevented the inhibition of Na+/H+ exchanger activity mediated by PTH-(28-48) and PMA but did not avert the negative regulation caused by PTH-(1-34) or forskolin. However, inhibition of both PKA and PKC prevented PTH-(1-34)-mediated suppression of Na+/H+ exchanger activity, indicating that PTH-(1-34) acted through both signaling pathways. In addition, Northern blot analysis revealed the presence of only the NHE-3 isoform of the Na+/H+ exchanger in OK cells. In summary, these results demonstrated that NHE-3 is expressed in OK cells and that activation of the PTH receptor can stimulate both the PKA and PKC pathways, each of which can independently lead to inhibition of NHE-3 activity.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and physical and genetic mapping of a novel human Na+/H+ exchanger (NHE5/SLC9A5) to chromosome 16q22.1.

A human genomic clone for a novel fifth member of the Na+/H+ exchanger (NHE) family, NHE5 (gene symbol SLC9A5), has been isolated and partially sequenced. The deduced amino acid sequence of two exons, containing 154 codons, exhibits 59-73% identity to the other members of the NHE family, with closest similarity to NHE3. Northern blot analysis demonstrated that the NHE5 gene is expressed in brain, testis, spleen, and skeletal muscle. Fluorescence in situ hybridization analysis of a cosmid containing NHE5 to human metaphase chromosomes localized the NHE5 gene to the cytogenetic interval 16q21-q22. A panel of somatic cell hybrids containing various portions of chromosome 16 was used to refine further the placement of NHE5 within band 16q22.1. A polymorphic dinucleotide (GT/CA)n repeat contained in the NHE5 cosmid was identified and developed into a microsatellite PCR marker. This was typed in a subset of the CEPH (Centre d'Etude du Polymorphisme Humain) families to place it on a genetic map of the human genome. Pairwise linkage analysis of this marker showed that it was linked to marker D16S421 with a maximal lod score of 35.21 at a recombination fraction (theta) of 0.000, in complete concordance with its chromosomal localization by physical mapping. Multipoint linkage analysis placed NHE5 between the flanking markers D16S421 and D16S512. The cloning of this new member of the sodium hydrogen exchanger family, its chromosomal localization, and the discovery of a polymorphic marker for it now make it feasible to study the possible involvement of this gene in disorders of Na+/H+ transport.

Amino Acid Sequence↗

PubMed1995

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

Animals↗

PubMed1995

Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress.

Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about the underlying mechanism. We probed the osmotic responsiveness of full-length and truncated constructs of NHE1 transfected into cells lacking endogenous antiport activity. The hypertonic stimulation of NHE1 was preserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants truncated at position 635 (delta 635) failed to respond to osmotic challenge. Transfectants (delta 635) behaved as if constitutively activated, having a permanently elevated cytosolic pH (pHi) under isotonic, unstimulated conditions. The delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs delta 582 or delta 566 had a reduced H+ sensitivity and were therefore inactive at resting pHi. Such cells were unresponsive to osmotic stress near physiological pHi but could be activated by shrinking after an acid load. Jointly, these results suggest that the H+ affinity and high cooperativity of the antiporter, earlier attributed to a single "modifier site," can be varied independently and are probably controlled by different regions of the molecule. The data indicate that volume or osmolarity-sensitive site(s) exist between the NH2-terminus and residue 566. This putative volume-sensitive site is therefore different from the site(s) postulated to mediate the stimulatory effects of calcium and growth factors.

Animals↗

PubMed1995

Role of glucocorticoids in the maturation of renal cortical Na+/H+ exchanger activity during fetal life in sheep.

We have studied the role of glucocorticoids in inducing the maturation in activity of the proximal tubule Na+/H+ exchanger that follows birth. Renal cortical microvillus membrane vesicles were prepared from 132-day gestation sheep fetuses (n = 8) that had received intraperitoneal cortisol (13 micrograms.kg-1.h-1) for the previous 48 h. Membrane vesicles were also obtained from sham-operated twin controls (n = 8). Amiloride-sensitive uptake of 22Na+ by these vesicles was measured, and Woolf-Augustinsson-Hofstee plots were used to determine the Michaelis constant (Km) and maximal velocity (Vmax). There was no significant difference in Km; however, the Vmax was 61% higher in cortisol-treated fetuses. Posttreatment circulating cortisol levels were significantly higher in the treated fetuses. Total RNA was collected from renal cortex of the eight pairs of twins when killed. Renal cortex Na+/H+ exchanger 3 (NHE3) mRNA levels were approximately fourfold higher in cortisol-treated than in control fetuses. Although proximal tubule Na+/H+ exchanger activity and renal cortex NHE3 mRNA levels increased significantly in cortisol-treated fetuses, cortisol infusion did not stimulate renal sodium reabsorption in the fetus but rather produced a natriuresis. These results demonstrate that glucocorticoids can induce an increase in both Na+/H+ exchanger activity and NHE3 mRNA levels during the last trimester of gestation in sheep. However, these changes are not associated with an increased ability of the fetal kidney to reabsorb sodium.

Animals↗

PubMed1995

Hemodialysis removal of acyclovir.

A 59-y-old with a history of chronic renal failure on hemodialysis was diagnosed with herpes zoster and begun on 800 mg acyclovir 5 times daily. Two days later the patient developed visual hallucinations, ataxia, confusion and memory loss along with focal myoclonus, nausea and vomiting. No fever, elevated WBC count or significant electrolyte imbalance was found. CT scan of the brain was unremarkable. The patient was then dialyzed for presumed acyclovir toxicity. Her acyclovir level was later found to have been 3.4 micrograms/ml (normal peak range 0.4-2 micrograms/ml) prior to dialysis. After 3 h of hemodialysis, her post-dialysis acyclovir level was 1.9 micrograms/ml. After a second course of hemodialysis the next day the patient's mental status improved, and she was discharged 5 d later. Due to its low volume of distribution (0.6 L/kg), low protein binding (about 15%) and water solubility, acyclovir is an example of the ideal drug that can be removed by hemodialysis. About 45% of the total body amount can be extracted through a 3-h course of hemodialysis with resultant improvement in symptoms.

Acyclovir↗

PubMed1994

Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation.

Four distinct isoforms of the mammalian Na+/H+ exchanger (NHE) have been identified by molecular cloning. Three of these (NHE-1, NHE-2, and NHE-3) have been shown to be functionally active by heterologous expression. Their kinetic and pharmacological properties are well documented, yet comparatively little is known about their regulation. In this report, rat NHE-1, NHE-2, and NHE-3 were stably transfected into antiporter-deficient Chinese hamster ovary cells to study their role in cellular proliferation and their regulation by nucleotides and cell volume. Their ability to influence cell proliferation was assessed by measuring the growth of antiporter-deficient cells and of the different transfectants in media of varying pH. While antiporter-deficient cells were unable to grow at acidic pH levels, all three isoforms supported proliferation under these conditions. Therefore, while the epithelia-specific isoforms (NHE-2 and NHE-3) are thought to play primarily a role in transcellular ion transport, they can also contribute to intracellular pH (pHi) homeostasis and have a permissive role in cell growth. The activity of the three isoforms was markedly inhibited by depletion of cellular ATP. In the pHi 6.0-7.2 range, decreases in the affinity for internal H+ and/or the maximal rate of transport accounted for the inhibitory effect, depending on the isoform. The osmotic responsiveness of the three isoforms was also compared. As reported earlier, NHE-1 was stimulated by hypertonicity. Under similar conditions, NHE-2 was also stimulated to a comparable extent. Conversely, both isoforms were inhibited in hypotonic media. In contrast, NHE-3 was markedly inhibited by hypertonic cell shrinking but was unaffected by hypotonicity. Osmotic inhibition of NHE-3 was rapid, reversible, and apparent throughout the pH range studied. Osmotic inhibition of NHE-3 may play a role in the physiology and pathophysiology of epithelia.

Adenosine Triphosphate↗

PubMed1994

Alteration of Na,K-ATPase subunit mRNA and protein levels in hypertrophied rat heart.

To determine if an altered expression of the Na,K-ATPase alpha isoform genes is responsible for an observed increase in cardiac glycoside sensitivity in compensatory hypertrophy, we performed Northern and slot blot analyses of RNA and specific immunological detection of Na,K-ATPase isoforms in rat hearts from normal and pressure overload-treated animals induced by abdominal aortic constriction. During the early phase of hypertrophy, the only alteration is a decrease in the alpha 2 mRNA isoform. In the compensated hypertrophied heart, the levels of the predominant alpha 1 isoform (mRNA and protein) and the beta 1 subunit mRNA are unchanged. In contrast, the alpha 2 isoform (mRNA and protein) is decreased by 35% and up to 61-64% in mild (< 55%) and severe (> 55%) hypertrophy, respectively. The alpha 3 isoform (mRNA and protein), which is extremely low in adult heart, is increased up to 2-fold during hypertrophy but accounts for only approximately equal to 5% of the total alpha isoform mRNA. These findings demonstrate that, in cardiac hypertrophy, the three alpha isoforms of the Na,K-ATPase are independently regulated and that regulation occurs at a pretranslational level. The pattern of expression in hypertrophied adult heart is similar to that of the neonatal heart where the inverse regulation between the alpha 2 and alpha 3 ouabain high affinity isoforms has been reported. This suggests that distinct regulatory mechanisms controlling Na,K-ATPase isoform expression may, at least in part, be involved in the sensitivity to cardiac glycosides.

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