The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.

Angiotensin II (AII) plays an important role in renal proximal tubular acidification via the costimulation of basolateral Na/HCO3 cotransporter (NBC) and apical Na/H exchanger (NHE) activities. These effects are mediated by specific G protein-coupled AII receptors, but their corresponding downstream effectors are incompletely defined. Src family tyrosine kinases (SFKs) contribute to the regulation of both transport activities by a variety of stimuli and are coupled to classic mitogen-activated protein kinase (MAPK) pathway activation in this cell type. We therefore examined these signaling intermediates for involvement in AII-stimulated NBC activity in cultured proximal tubule cells. Subpressor concentrations of AII (0.1 nM) increased NBC activity within minutes, and this effect was abrogated by selective antagonism of AT1 angiotensin receptors, SFKs, or the classic MAPK pathway. AII directly activated Src, as well as the proximal (Raf) and distal (ERK) elements of the classic MAPK module, and the activation of Src was prevented by AT1 receptor antagonism. An associated increase in basolateral membrane NBC1 content is compatible with the involvement of this proximal tubule isoform in these changes. We conclude that AII stimulation of the AT1 receptor increases NBC activity via sequential activation of SFKs and the classic MAPK pathway. Similar requirements for SFK/MAPK coupling in both cholinergic and acidotic costimulation of NBC and NHE activities suggest a central role for these effectors in the coordinated regulation of epithelial transport by diverse stimuli.

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-...

We have previously shown that the activity of the Na-HCO3 cotransporter is stimulated by protein kinase C (PKC) activation, but the mechanism responsible for this effect is not clear. We have shown that cultured proximal tubule cells of the rabbit have DIDS-sensitive Na-HCO3 cotransporter activity as assessed by HCO3-dependent 22Na uptake or by measurement of intracellular pH. In cells loaded with BCECF and treated with the amiloride analogue, ethylisopropyl amiloride, removal of extracellular Na was associated with a rapid decrease in pH which returned to normal with re-addition of Na. This pH recovery was inhibited by DIDS and was used to quantify the activity of the Na-HCO3 cotransporter. In the present study, we utilized primary cultures of the proximal tubule of the rabbit to examine the effect of PKC activation on the activity of the Na-HCO3 cotransporter. Short term incubation (5 min) with the active phorbol ester, phorbol 12-myristate, 13-acetate (PMA), 10(-7) M, caused a significant stimulation of the Na-HCO3 cotransporter activity as compared to controls. Incubation for two hours also caused a significant stimulation of the Na-HCO3 cotransporter activity. The inactive analogue of PMA, 4-alpha phorbol, failed to alter the cotransporter. Similar results were observed when we examined the effect of PMA on HCO3-dependent 22Na uptake. The effect of PMA to stimulate the cotransporter was mediated by PKC activation since it could be prevented by the PKC inhibitors, calphostin C or sphingosine, or by prior PKC depletion. The long term but not the short term effect of PMA to stimulate the Na-HCO3 cotransporter activity was prevented by the protein synthesis inhibitors, actinomycin D or cycloheximide. The early effect of PKC to stimulate the cotransporter appeared to be associated with increased phosphorylation of a 56 kD protein band, while the late effect appeared to be associated with an increase in immunoreactive content of a 56 kD protein which is thought to be an active component of the cotransporter. Thus PKC stimulation activates the Na-HCO3 cotransporter by two distinct mechanisms: a long term effect which is protein synthesis-dependent and a short term effect which is protein synthesis-independent and is likely mediated by phosphorylation.

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP-PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na+/HCO3- cotransporter. We investigated whether the activity of Na+/HCO3- cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 micrograms) increased 22Na uptake in the presence of HCO3 but not in the presence of gluconate, indicating that trypsin does not alter diffusive 22Na uptake but directly stimulates the Na+/HCO3- cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na+/HCO3- cotransporter (expressed as nanomoles/mg protein/3s) from 23 +/- 10 to 14 +/- 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na+/HCO3- cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na+/HCO3- cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na+/HCO3- cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S-).(ABSTRACT TRUNCATED AT 250 WORDS)