Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Hanai, Jun‐ichi; Takaichi, Kenmei; Miyajima, Yoshihiro; Fujita, Toshiro; Kurokawa, Kiyoshi

doi:10.1002/(sici)1097-4652(199706)171:3<318::aid-jcp10>3.0.co;2-6

“…Activities of the different NHE isoforms are affected by PKC-and PKA-dependent signalling pathways (Kandasamy et al, 1995), and the exchangers might therefore be involved in regulation of D-Phe-L-Ala transport activity. LLC-PK 1 cells were found to express NHE-3 in the apical (Soleimani et al, 1994) and NHE-1 in the basolateral membrane (Hanai et al, 1997). When LLC-PK 1 cells were preincubated with staurosporine at pH 7.4, the subsequent pH in decline induced by superfusion with pH 6.0 buffer was significantly larger than that in control cells preincubated in the absence of the PKC inhibitor ( Fig.…”

Section: Alterations Of the Transmembrane H ؉ -Gradientmentioning

confidence: 87%

“…Consequently, any regulation of peptide transport activity in LLC-PK 1 cells could be secondary to changes in the proton gra- dient and the driving force maintained by NHE transporters. The LLC-PK 1 cell line has been described to express the epithelial NHE-3 isoform in the apical (Soleimani et al, 1994) and the housekeeping NHE-1 isoform in the basolateral membrane (Hanai et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Regulation of the high‐affinity H/peptide cotransporter in renal LLC‐PK1 cells

Wenzel

¹

,

Diehl

²

,

Herget

³

et al. 1999

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Di- and tripeptides and peptide mimetics such as beta-lactam antibiotics are efficiently reabsorbed from the tubular lumen by a high-affinity peptide transporter. We have recently identified and characterized this H+-coupled high-affinity peptide transport system in the porcine proximal tubular cell line LLC-PK1. Here we describe for the first time the regulation of the renal high-affinity peptide cotransporter at the cellular level. Uptake of 5 microM 3H-D-Phe-L-Ala into LLC-PK1 cells was significantly increased by lowering [Ca2+]in and decreased by increasing [Ca2+] in. Moreover, it was shown that the [Ca2+]in effects on peptide transport activity were dependent on Ca2+ entry from the extracellular site (e.g., via a store-regulated capacitative Ca2+ influx). Protein kinase C (PKC) was found to transmit the effects of [Ca2+]in on peptide transport. Although we demonstrate by pHin measurements that the PKC inhibitor staurosporine did decrease the transmembrane H+ gradient and consequently should have reduced the driving force for peptide uptake, the only effect on transport kinetics of 3H-D-Phe-L-Ala observed was a significant decrease in Km from 22.7+/-2.5 microM to 10.2+/-1.9 microM with no change in maximal velocity.

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Expression of transfected human Na+/H+ exchanger (NHE-1) in the basolateral membrane of opossum kidney cells

Takaichi

¹

,

Miyajima

²

,

Hanai

³

et al. 1999

J. Cell. Physiol.

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Some epithelial cells have Na+/H+ exchanger (NHE) activity in both apical and basolateral membranes. Amiloride-sensitive NHE-1 is generally identified in the basolateral membrane. The renal cell line, OK7a, targets amiloride-resistant NHE predominantly to the apical membrane. It is controversial whether the transfected NHE-1 is targeted preferentially to the basolateral membrane in OK7a cells, when human NHE-1 is chronically expressed under control of constitutively active promoters. We tried to identify the membranes in which the transfected human NHE-1 could be detected following acute expression in OK7a cells. We have always observed small Na(+)-dependent pH recovery in the basolateral membrane in OK7a cells. It is, however, controversial whether or not OK7a cells express NHE activity in the basolateral membrane. We also characterized Na(+)-dependent pH recovery in the basolateral membrane. It was not inhibited by [4,4'diisothiocyanatostilbene-2,2'-disulfonic acid] (DIDS), [4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid] (SITS), or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These results are consistent with the presence of the NHE in the basolateral membrane. NHE activities were predominant in the apical membrane and those in both membranes were resistant to amiloride analogs. After stable transfection with human NHE-1 in a vector utilizing the metallothionein promoter, overnight induction with Zn(2+)increased the NHE activity and its sensitivity to amiloride only in the basolateral membrane in OK7a cells. We conclude that the transfected human NHE-1 is exclusively targeted to the basolateral membrane of OK7a cells during acute induction.

show abstract

Regulation of the high-affinity H+/peptide cotransporter in renal LLC-PK1 cells

Wenzel

¹

,

Diehl

²

,

Herget

³

et al. 1999

View full text Add to dashboard Cite

Di- and tripeptides and peptide mimetics such as beta-lactam antibiotics are efficiently reabsorbed from the tubular lumen by a high-affinity peptide transporter. We have recently identified and characterized this H+-coupled high-affinity peptide transport system in the porcine proximal tubular cell line LLC-PK1. Here we describe for the first time the regulation of the renal high-affinity peptide cotransporter at the cellular level. Uptake of 5 microM 3H-D-Phe-L-Ala into LLC-PK1 cells was significantly increased by lowering [Ca2+]in and decreased by increasing [Ca2+] in. Moreover, it was shown that the [Ca2+]in effects on peptide transport activity were dependent on Ca2+ entry from the extracellular site (e.g., via a store-regulated capacitative Ca2+ influx). Protein kinase C (PKC) was found to transmit the effects of [Ca2+]in on peptide transport. Although we demonstrate by pHin measurements that the PKC inhibitor staurosporine did decrease the transmembrane H+ gradient and consequently should have reduced the driving force for peptide uptake, the only effect on transport kinetics of 3H-D-Phe-L-Ala observed was a significant decrease in Km from 22.7+/-2.5 microM to 10.2+/-1.9 microM with no change in maximal velocity.

show abstract

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Cited by 4 publications

References 32 publications

Regulation of the high‐affinity H/peptide cotransporter in renal LLC‐PK1 cells

Regulation of the high‐affinity H/peptide cotransporter in renal LLC‐PK1 cells

Expression of transfected human Na+/H+ exchanger (NHE-1) in the basolateral membrane of opossum kidney cells

Regulation of the high-affinity H+/peptide cotransporter in renal LLC-PK1 cells

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