Renal cortical basolateral Na+/HCO 3 ? cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

Bernardo, Angelito; Kear, F.T.; Stim, James; Qiu, Yi-Yong; Ruiz, Ofelia S.; Weidman, H.; Arruda, José A.L.

doi:10.1007/bf00233307

Cited by 6 publications

(9 citation statements)

References 18 publications

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“…However, we have previously shown that limited trypsin digestion of solubilized renal BLMs is sufficient to dissociate NBC activity from its regulation by PKA (13). Co-reconstitution experiments have further suggested that a regulatory protein analogous to NHE-RF is necessary for NBC regulation by PKA (13). Taken together, these findings suggest a role for NHE-RF in PKA regulation of NBC activity.…”

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confidence: 87%

“…BLMs and BBMs were prepared from rabbit kidney cortex as described previously (8,13,14). BBM purity was assessed by enrichment of alkaline phosphatase activity, and specific activity was typically 10-to 14-fold greater than that of homogenates.…”

Section: Preparation Of Membrane Vesicles and Proteoliposomesmentioning

confidence: 99%

“…BLM preparations were similarly 12-to 15-fold increased in Na + /K + /ATPase content, without a corresponding increase in the specific activities of luminal membranes markers (e.g., alkaline phosphatase and γ-glutamyl transferase). BLM and BBM proteins were solubilized and reconstituted into proteoliposomes as described (13,15,16). In brief, liposomal reconstitution of solubilized proteins (2.5 mg/mL) was accomplished by mixing 1.6 part protein (vol/vol) with 1.0 part of L-α phosphatidylcholine (35 mg/mL) and sonicating at maximal output and 4°C for 10 minutes.…”

Section: Preparation Of Membrane Vesicles and Proteoliposomesmentioning

confidence: 99%

“…Initial rates of bicarbonate-dependent 22 Na uptake were used to measure maximal NBC activity in proteoliposomes and were measured at 3 seconds by the rapid-filtration technique, as described previously and outlined later here (13,15). After preincubation at room temperature for 1-2 hours in 200 mM sucrose, 1 mM gluconate, and 50 mM Tris-buffered HEPES (pH 7.5), proteoliposomes were pelleted at 30,000 g for 30 minutes at 4°C before resuspension in the same solution.…”

Section: Preparation Of Membrane Vesicles and Proteoliposomesmentioning

confidence: 99%

“…This has not yet been demonstrated for the NBC and thus requires further investigation. However, we have previously shown that limited trypsin digestion of solubilized renal BLMs is sufficient to dissociate NBC activity from its regulation by PKA (13). Co-reconstitution experiments have further suggested that a regulatory protein analogous to NHE-RF is necessary for NBC regulation by PKA (13).…”

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confidence: 99%

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Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

Bernardo¹,

Kear²,

Santos³

et al. 1999

J. Clin. Invest.

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IntroductionThe renal proximal tubule reabsorbs more than 80% of the filtered bicarbonate load. The principal transport systems (1-3) responsible for transmembrane movement of hydrogen and bicarbonate ions in the proximal tubule cells are the apical Na + /H -exchanger type 3 isoform (NHE3) and the basolateral Na + /HCO 3 -cotransporter (NBC). It might be predicted that systemic and proximal tubule intracellular acid-base homeostasis requires that NHE3 and NBC function in a coordinate fashion. The uniformly parallel regulation of both transporters under a variety of physiologic conditions is consistent with such a hypothesis. NBC activity is enhanced by metabolic acidosis and inhibited by metabolic alkalosis (4). Similarly, chronic hypercapnia increases NBC activity, whereas chronic respiratory alkalosis has the opposite effect (5). NBC activity is also modulated by angiotensin II and parathyroid hormone (6, 7). Under identical conditions, NHE3 activity varies in parallel, suggesting that the activities of the 2 systems may be coordinately regulated (4). The parallel regulation of these transporters has also recently been extended to the level of specific regulatory protein kinases (8, 9). Phosphorylation of brush-border membrane (BBM) and basolateral membrane (BLM) proteins by either protein kinase A (PKA) or calcium calmodulin multifunction protein kinase II (Ca-CAMK II) is inhibitory for both NHE and NBC activities in the respective membranes, whereas protein kinase C (PKC) is stimulatory (8). In the renal proximal tubule, the activities of the basolateral Na + /HCO 3 -cotransporter (NBC) and the apical Na + /H + exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na + /H + exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10 -8 M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-...

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confidence: 87%

Section: Preparation Of Membrane Vesicles and Proteoliposomesmentioning

confidence: 99%

Section: Preparation Of Membrane Vesicles and Proteoliposomesmentioning

confidence: 99%

Section: Preparation Of Membrane Vesicles and Proteoliposomesmentioning

confidence: 99%

mentioning

confidence: 99%

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Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

Bernardo¹,

Kear²,

Santos³

et al. 1999

J. Clin. Invest.

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Renal Cortical Basolateral Na + /HCO − 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

Bernardo

Kear

Stim

et al. 1996

Journal of Membrane Biology

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We have previously partially purified the basolateral Na+/HCO3- cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO3- cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na+/ HCO3- cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO3- cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO3- cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO3- cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na+/HCO3- cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na+/HCO3- cotransporter protein is expressed in different regions of the kidneys and in other tissues.

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The Renal Cortical Na + /HCO 3 − Cotransporter VI: The Effect of Chemical Modification in Cotransporter Activity

Bernardo

Kear

Arruda

1997

Journal of Membrane Biology

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The Na+/HCO3- cotransporter is the main system that mediates bicarbonate removal out of the proximal tubule cell into the blood. We have previously partially purified this protein and showed that chemical modification of the alpha-amino groups by fluorescein isothiocyanate (FITC) inhibited the activity of the Na+/HCO3- cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that this compound binds at or near the substrate transport sites of the cotransporter. We examined the effect of agents that modify the sulfhydryl group (dithiothreitol), carboxyl groups (n-n'dicyclohexyl carbodiimide) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which may be important for transport function. The sulfhydryl residues modifier, carboxyl group modifier, and tyrosine modifier significantly inhibited bicarbonate dependent 22Na uptake in basolateral membranes by 50-70% without altering the 22Na uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting that the tyrosine residues are not at the substrate binding sites. To determine the presence and role of glycosylation on the Na+/HCO3- cotransporter protein, we examined the effects of different glycosidases (endoglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransporter activity. All glycosidases caused a significant 50-80% inhibition of cotransporter activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na+/HCO3- cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl groups as well as glycosylation are important for expression of full functional activity of the cotransporter.

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Renal cortical basolateral Na+/HCO 3 ? cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

Cited by 6 publications

References 18 publications

Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

Renal Cortical Basolateral Na + /HCO − 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

The Renal Cortical Na + /HCO 3 − Cotransporter VI: The Effect of Chemical Modification in Cotransporter Activity

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