Evaluation of the toxicity of zinc in the rat olfactory neuronal cell line, Odora.

Zinc (Zn) has long been touted as a panacea for common cold. Recently, there has been some controversy over whether an intranasal (IN) zinc gluconate gel, purported to fight colds, causes anosmia, or loss of the sense of smell, in humans. Previous evidence has shown that IN zinc sulfate (ZnSO4) solutions can cause anosmia in humans as well as significant damage to the olfactory epithelium in rodents. Using an in vitro olfactory neuron model (the rat Odora cell line), we tested the hypothesis that Zn toxicity was caused by inhibition of the hydrogen voltage-gated channel 1(HVCN1), leading to acidosis and apoptotic cell death. Following studies to characterize the toxicity of zinc gluconate and ZnSO4, Odora cells were grown on coverslips and loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH in the presence and absence of Zn salts. While we found that HVCN1 is not functional in Odora cells, we found that olfactory neurons in vitro maintain their intracellular pH through a sodium/proton exchanger, specifically the sodium proton antiporter 1. ZnSO4, at nontoxic levels, had no impact on intracellular pH after acute exposure or after 24 h of incubation with the cells. In conclusion, Zn toxicity is not mediated through an acidification of intracellular pH in olfactory neurons in vitro.

Estrogen downregulates the proximal tubule type IIa sodium phosphate cotransporter causing phosphate wasting and hypophosphatemia.

Estrogen treatment causes significant hypophosphatemia in patients. To determine the mechanisms responsible for this effect, we injected ovariectomized rats with either 17beta-estradiol or vehicle for three days. Significant renal phosphate wasting and hypophosphatemia occurred in estrogen-treated rats despite a decrease in their food intake. The mRNA and protein levels of the renal proximal tubule sodium phosphate cotransporter (NaPi-IIa) were significantly decreased in estradiol-treated ad-libitum or pair-fed groups. Estrogen did not affect NaPi-III or NaPi-IIc expression. In ovariectomized and parathyroidectomized rats, 17beta-estradiol caused a significant decrease in NaPi-IIa mRNA and protein expression compared to vehicle. Estrogen receptor alpha isoform blocker significantly blunted the anorexic effect of 17beta-estradiol but did not affect the downregulation of NaPi-IIa. Our studies show that renal phosphate wasting and hypophosphatemia induced by estrogen are secondary to downregulation of NaPi-IIa in the proximal tubule. These effects are independent of food intake or parathyroid hormone levels and likely not mediated through the activation of estrogen receptor alpha subtype.

Coordinated down-regulation of NBC-1 and NHE-3 in sodium and bicarbonate loading.

BACKGROUND: Bicarbonate reabsorption in the kidney proximal tubule is predominantly mediated via the apical Na+/H+ exchanger (NHE-3) and basolateral Na+: HCO(-3) cotransporter (NBC-1). The purpose of these studies was to examine the effects of Na+ load and altered acid-base status on the expression of NHE-3 and NBC-1 in the kidney. METHODS: Rats were placed on 280 mmol/L of NaHCO(3), NaCl, or NH(4)Cl added to their drinking water for 5 days and examined for the expression of NHE-3 and NBC-1 in the kidney. RESULTS: Serum [HCO(-3)] was unchanged in NaHCO(-3) and NaCl-loaded animals versus control (P> 0.05). However, a significant hyperchloremic metabolic acidosis was developed in NH4Cl-loaded animals. A specific polyclonal antibody against NBC-1 recognized a 130 kD band, which was exclusively expressed in the basolateral membrane of proximal tubules. Immunoblot studies indicated that the protein abundance of NBC-1 and NHE-3 in the cortex decreased by 74% (P < 0.04) and 66% (P < 0.03), respectively, in NaHCO(3) loading and by 72% (P < 0.003) and 55% (P < 0.04), respectively, in NaCl loading. Switching from NaHCO(3) to distilled water resulted in rapid recovery of NHE-3 and NBC-1 protein expression toward normal levels. Metabolic acidosis increased the abundance of NHE-3 (P < 0.0001) but not NBC-1 (P> 0.05). CONCLUSIONS: NaHCO(-3) or NaCl loading coordinately down-regulates the apical NHE-3 and basolateral NBC-1 in rat kidney proximal tubule, presumably due to increased Na+ load. We propose that the down-regulation of these two Na+- and HCO(3)-absorbing transporters is, to a large degree, responsible for enhanced excretion of excess of Na+ and alkaline load and prevention of metabolic alkalosis in rats subjected to NaHCO(-3) loading.

Mouse Na+: HCO3- cotransporter isoform NBC-3 (kNBC-3): cloning, expression, and renal distribution.

BACKGROUND: Na+:HCO3- cotransporters mediate the transport of HCO3- into or out of the cell. We recently reported the partial cloning and characterization of a new human Na+:HCO3- cotransporter (referred to as NBC-3 or kNBC-3). The purpose of the present studies was to clone the mouse kNBC-3 and to examine its properties and expression in the kidney. METHODS: Using primers from human kNBC-3 cDNA and 5' and 3' rapid amplification cDNA end polymerase chain reaction (RACE PCR), the mouse kNBC-3 full-length cDNA was cloned from inner medullary collecting duct (mIMCD-3) cells. The tissue distribution and functional properties of NBC-3 was determined using established methods. RESULTS: The coding region of the mouse kNBC-3 has 1089 amino acids and shows 73 and 56% identity to human NBC-2 and NBC-1, respectively. The renal distribution of kNBC-3 demonstrated a unique expression pattern: Whereas kNBC-1 is predominantly expressed in the cortex and is absent in the inner medulla, kNBC-3 shows an intense expression level in the inner medulla and is absent in the cortex. Expression studies in oocytes indicated that NBC-3 mediates Na-dependent HCO3- cotransport. Electrophysiological experiments demonstrated that unlike kNBC-1, which is electrogenic, kNBC-3 is electroneutral. CONCLUSIONS: Based on its distribution and electroneutrality, we propose that kNBC-3 mediates the transport of HCO3- into the cells.

Pendrin: an apical Cl-/OH-/HCO3- exchanger in the kidney cortex.

The identities of the apical Cl-/base exchangers in kidney proximal tubule and cortical collecting duct (CCD) cells remain unknown. Pendrin (PDS), which is expressed at high levels in the thyroid and its mutation causes Pendred's syndrome, is shown to be an anion exchanger. We investigated the renal distribution of PDS and its function. Our results demonstrate that pendrin mRNA expression in the rat kidney is abundant and limited to the cortex. Proximal tubule suspensions isolated from kidney cortex were highly enriched in pendrin mRNA. Immunoblot analysis studies localized pendrin to cortical brush-border membranes. Nephron segment RT-PCR localized pendrin mRNA to proximal tubule and CCD. Expression studies in HEK-293 cells demonstrated that pendrin functions in the Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange modes. The conclusion is that pendrin is an apical Cl-/base exchanger in the kidney proximal tubule and CCD and mediates Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange.

Fasting downregulates renal water channel AQP2 and causes polyuria.

Starvation causes impairment in the urinary concentrating ability. The mechanism of this defect, however, remains unknown. We tested the possibility that food deprivation might affect the expression and activity of aquaporins (AQP1, 2), thereby impairing renal water reabsorption in the kidney. Rats fasted for 24 h exhibited severe polyuria (urine volume increased from 11 before fasting to 29 ml/24 h after fasting, P < 0.0001) along with failure to concentrate their urine (urine osmolality decreased from 1,485 before fasting to 495 mosmol/kgH(2)O after fasting, P < 0.0001). Refeeding for 24 h returned the urinary concentrating ability back to normal. Northern hybridization and immunoblot analysis demonstrated that fasting was associated with a decrease in AQP2 protein (-80%, P

Ammonium carriers in medullary thick ascending limb.

Absorption of NH(4)(+) by the medullary thick ascending limb (MTAL) is a key event in the renal handling of NH(4)(+), leading to accumulation of NH(4)(+)/NH(3) in the renal medulla, which favors NH(4)(+) secretion in medullary collecting ducts and excretion in urine. The Na(+)-K(+)(NH(4)(+))-2Cl(-) cotransporter (BSC1/NKCC2) ensures approximately 50-65% of MTAL active luminal NH(4)(+) uptake under basal conditions. Apical barium- and verapamil-sensitive K(+)/NH(4)(+) antiport and amiloride-sensitive NH(4)(+) conductance account for the rest of active luminal NH(4)(+) transport. The presence of a K(+)/NH(4)(+) antiport besides BSC1 allows NH(4)(+) and NaCl absorption by MTAL to be independently regulated by vasopressin. At the basolateral step, the roles of NH(3) diffusion coupled to Na(+)/H(+) exchange or Na(+)/NH(4)(+) exchange, which favors NH(4)(+) absorption, and of Na(+)/K(+)(NH(4)(+))-ATPase, NH(4)(+)-Cl(-) cotransport, and NH(4)(+) conductance, which oppose NH(4)(+) absorption, have not been quantitatively defined. The increased ability of the MTAL to absorb NH(4)(+) during chronic metabolic acidosis involves an increase in BSC1 expression, but fine regulation of MTAL NH(4)(+) transport probably requires coordinated effects on various apical and basolateral MTAL carriers.

Pathways for HCO3-reabsorption in mouse medullary collecting duct segments.

To determine pathways of HCO3- reabsorption in the collecting duct of the mouse kidney, the outer medullary collecting duct (OMCD) and the terminal inner medullary collecting duct (IMCDt) were dissected and perfused at 1 to 2 nL/min, and total CO2 was measured by microfluorometry. In the OMCD, net HCO3- flux (JtCO2) was 12.2 +/- 0.7 pmol/min/mm tubule length and decreased to 6.9 +/- 0.6 pmol/min/mm tubule length (n = 5) with 10 mol/L Schering 28080 (SCH) in perfusate (P < .001) and to 7.7 +/- 0.6 pmol/min/mm tubule length (P < .004; n = 4) with 50 micromol/L diethylstilbestrol (DES), an inhibitor of H+-adenosine triphosphatase; together they reduced JtCO2 to 3.7 +/- 0.2 pmol/min/mm tubule length (P = .0002; n = 4). In IMCDt, JtCO2 was 10.9 +/- 1.1 pmol/min/mm tubule length, and it decreased to 4.3 +/- 0.9 pmol/min/mm tubule length (n = 4) with 10 micromol/L SCH in perfusate (P < .05) and to 7.0 +/- 1.1 pmol/min/mm tubule length (P < .05; n = 4) with 50 micromol/L DES; together they decreased JtCO2 to 2.3 +/- 0.3 pmol/min/mm tubule length (P < .002; n = 4). Ouabain (1 mmol/L), an inhibitor of colonic H-K-adenosine triphosphatase (cHKA), in perfusate had no effect on JtCO2 in either segment. Northern hybridization studies showed a high level of expression of gastric HKA (gHKA) in outer medulla and a low level in inner medulla; cHKA expression was undetectable. Thus, in normal mouse OMCD and IMCDt, HCO3- reabsorption is predominantly mediated by gHKA and H+-adenosine triphosphatase and not cHKA. A third isoform of HKA could be present in mouse IMCDt.

Early polyuria and urinary concentrating defect in potassium deprivation.

The time course of the onset of nephrogenic diabetes insipidus and its relationship to aquaporin-2 (AQP2) expression in K(+) deprivation (KD) remains unknown. Rats were fed a K(+)-free diet and killed after 12 h, 1, 2, 3, 6, or 21 days. Serum K(+) concentration was decreased only after, but not before, 3 days of a K(+)-free diet. Urine osmolality, however, decreased as early as 12 h of KD (1,061 +/- 26 vs. 1,487 +/- 102 mosmol/kgH(2)O in control, P < 0.01). It decreased further at 24 h (to 858 +/- 162 mosmol/kgH(2)O in KD, P < 0.004) and remained low at 21 days of KD (436 +/- 58 mosmol/kgH(2)O, P < 0.0001 compared with baseline). Water intake decreased at 12 h (P < 0.002) but increased at 24 h (P < 0.05) and remained elevated at 21 days of KD. Urine volume increased at 24 h of KD (8 +/- 2 to 15 +/- 2 ml/24 h, P < 0.05) and remained elevated at 21 days. Immunoblot analysis demonstrated that AQP2 protein abundance in the outer medulla remained unchanged at 12 h (P > 0.05), decreased at 24 h ( approximately 44%, P < 0.001), and remained suppressed ( approximately 52%, P < 0.03) at 21 days of KD. In the inner medulla the AQP2 protein abundance remained unchanged at both 12 and 24 h of KD. AQP2 protein abundance in the cortex, however, decreased at 12 h ( approximately 47%, P < 0.01) and remained suppressed at 24 h ( approximately 77%, P < 0.001) of KD. Northern blot analysis showed that AQP2 mRNA decreased as early as 12 h of KD in both cortex (P < 0.02) and outer medulla (P < 0.01) and remained suppressed afterward. In conclusion, the urinary concentrating defect in KD is an early event and precedes the onset of hypokalemia. These studies further suggest that the very early urinary concentrating defect in KD (after 12 but before 24 h) results primarily from the suppression of cortical AQP2, whereas the later onset of a urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2.

Potassium deprivation upregulates expression of renal basolateral Na(+)-HCO(3)(-) cotransporter (NBC-1).

The purpose of the present experiments was to examine the effect of potassium deprivation on the expression of the renal basolateral Na(+)-HCO(3)(-) cotransporter (NBC-1). Rats were placed on a K(+)-free diet for various time intervals and examined. NBC-1 mRNA levels increased by about threefold in the cortex (P < 0.04) at 72 h of K(+) deprivation and remained elevated at 21 days. NBC activity increased by approximately 110% in proximal tubule suspensions, with the activity increasing from 0.091 in control to 0.205 pH/min in the K(+)-deprived group (P < 0.005). The inner stripe of outer medulla and cells of medullary thick ascending limb of Henle (mTAL) showed induction of NBC-1 mRNA and activity in K(+)-deprived rats, with the activity in mTAL increasing from 0.010 in control to 0.133 pH/min in the K(+)-deprived group (P < 0.004). K(+) deprivation also increased NBC-1 mRNA levels in the renal papilla (P < 0.02). We conclude that 1) K(+) deprivation increases NBC-1 expression and activity in proximal tubule and 2) K(+) deprivation causes induction of NBC-1 expression and activity in mTAL tubule and inner medulla. We propose that NBC-1 likely mediates enhanced HCO(3)(-) reabsorption in proximal tubule, mTAL, and inner medullary collecting duct in K(+) deprivation and contributes to the maintenance of metabolic alkalosis in this condition.

Glucocorticoids enhance the expression of the basolateral Na+:HCO3- cotransporter in renal proximal tubules.

BACKGROUND: Studies have shown that glucocorticoids enhance HCO3- reabsorption in proximal tubules. Functional and molecular studies indicate that HCO3- reabsorption in proximal tubules is mediated via luminal H(+)-ATPase and Na+/H+ exchanger (NHE-3), and basolateral Na+:HCO3- cotransporter (NBC) acting in series. The purpose of these experiments was to examine the effect of adrenal steroids on NBC-1 and NHE-3 expression and activity in rat renal proximal tubules. METHODS: Rats were injected subcutaneously with dexamethasone (100 mu/day) or deoxycorticosterone acetate (30 mg/kg), potent glucocorticoid, or mineralocorticoid analogues, respectively. Animals were sacrificed after two or four days, and NBC-1 and NHE-3 mRNA expression and activities were measured in cortex and proximal tubules. RESULTS: Northern hybridizations indicated that cortical NBC-1 mRNA expression increased by approximately 92% in rats treated with dexamethasone for four days (N = 6, P < 0.03) but not two days. NHE-3 mRNA expression remained unchanged. NBC and NHE-3 activities were measured as the Na-dependent pHi recovery from an acid load in the presence or absence of HCO3-, respectively, and appropriate inhibitors in proximal tubule suspensions loaded with BCECF. NBC activity increased by approximately 80% in rats treated with dexamethasone for four days (P < 0.01, N = 5) but not two days. NHE-3 activity increased by 34 and 42% in rats treated with dexamethasone for two and four days, respectively (P < 0.05 and P < 0.02 for each group vs. control). Treatment with deoxycorticosterone acetate did not alter NBC-1 expression. CONCLUSION: Glucocorticoids at pharmacologic concentrations enhance the mRNA expression and functional activity of renal proximal tubule NBC-1. Enhanced NBC and NHE-3 activities could result in increased HCO3- reabsorption in proximal tubule and could contribute to the maintenance of metabolic alkalosis in pathophysiologic states associated with increased glucocorticoid production.

NH4+ secretion in inner medullary collecting duct in potassium deprivation: role of colonic H+-K+-ATPase.

UNLABELLED: NH4+ secretion in inner medullary collecting duct in potassium deprivation: Role of colonic H+-K+-ATPase. BACKGROUND: In K+ deprivation (KD), gastric (g) H+-K+-ATPase (HKA) is suppressed, whereas colonic (c) HKA is induced in the terminal inner medullary collecting duct (IMCD). We hypothesized that in KD, cHKA is induced and can mediate the secretion of NH4+. METHODS: Rats were sacrificed after 2, 3, 6, or 14 days on regular (NML) or K+-free (KD) diet. mRNA expression of HKA isoforms in terminal inner medulla was examined and correlated with NH4+ secretion in perfused IMCD in vitro. RESULTS: Urinary NH4+ excretion increased after K+-free diet for six days. In terminal inner medulla, cHKA expression was strongly induced, whereas gHKA expression was decreased. NH4+ secretion increased by 62% in KD (JtNH4+ 0.57 vs. 0.92 pmol/min/mm tubule length, P < 0.001). Ouabain (1 mM) in perfusate inhibited NH4+ secretion in KD by 45% (P < 0.002) but not in NML. At luminal pH 7.7, which inhibits NH3 diffusion, NH4+ secretion in IMCD was 140% higher in KD (0.36 vs. 0.15, P < 0.03) and was sensitive to ouabain. ROMK-1 mRNA expression was induced in parallel with cHKA in inner medulla. CONCLUSIONS: These data suggest that in KD, cHKA replaces gHKA and mediates enhanced secretion of NH4+ (and H+) into the lumen facilitated by K+ recycling through ROMK-1.

HCO-3 reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response.

Mice with a targeted disruption of Na+/H+ exchanger NHE-3 gene show significant reduction in HCO-3 reabsorption in proximal tubule, consistent with the absence of NHE-3. Serum HCO-3, however, is only mildly decreased (P. Schulties, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998), indicating possible adaptive upregulation of HCO-3-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 -/-) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2 (net HCO-3 flux, JtCO2) was measured in the presence of 10 microM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 microM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD, JtCO2 increased in NHE-3 mutant mice (3.42 +/- 0.28 in WT to 5.71 +/- 0.39 pmol. min-1. mm tubule-1 in mutants, P < 0.001). The SCH-sensitive net HCO-3 flux remained unchanged, whereas the DES-sensitive HCO-3 flux increased in the CCD of NHE-3 mutant animals. In OMCD, JtCO2 increased in NHE-3 mutant mice (8.8 +/- 0.7 in WT to 14.2 +/- 0.6 pmol. min-1. mm tubule-1 in mutants, P < 0.001). Both the SCH-sensitive and the DES-sensitive HCO-3 fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl-/HCO-3 exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate that HCO-3 reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatory HCO-3 reabsorption in NHE-3-deficient animals.

Characterization of Na+/HCO-3 cotransporter isoform NBC-3.

Na+-HCO-3 cotransporters mediate the transport of HCO-3 into or out of the cell. Two Na+-HCO-3 cotransporters (NBC) have been identified previously, which are referred to as NBC-1 and NBC-2. A cDNA library from uninduced human NT-2 cells was screened with an NBC-2 cDNA probe. Several clones were identified and isolated. Sequence analysis of these clones identified a partial coding region (2 kb) of a novel NBC (called here NBC-3), which showed 53% and 72% identity with NBC-1 and NBC-2, respectively. Northern blot analysis revealed that NBC-3 encodes a 4.4-kb mRNA with a tissue distribution pattern distinct from NBC-1 and NBC-2. NBC-3 is highly expressed in brain and spinal column, with moderate levels in trachea, thyroid, and kidney. In contrast with NBC-1, NBC-3 shows low levels of expression in pancreas and kidney cortex. In the kidney, NBC-3 expression is predominantly limited to the medulla. Cultured mouse inner medullary collecting duct (mIMCD-3) cells showed high levels of NBC-1 and low levels of NBC-3 mRNA expression. Subjecting the mutagenized mIMCD-3 cells to sublethal acid stress decreased the mRNA expression of NBC-1 by approximately 90% but increased the Na+-dependent HCO-3 cotransport activity by approximately 7-fold (as assayed by DIDS-sensitive, Na+-dependent, HCO-3-mediated intracellular pH recovery). This increase was associated with approximately 5.5-fold enhancement of NBC-3 mRNA levels. NBC showed significant affinity for Li+ in the mutant but not the parent mIMCD-3 cells. On the basis of the widespread distribution of NBC-3, we propose that this isoform is likely involved in cell pH regulation by transporting HCO-3 from blood to the cell. We further propose that enhanced expression of NBC-3 in severe acid stress could play an important role in cell survival by mediating the influx of HCO-3 into the cells.

CFTR drives Na+-nHCO-3 cotransport in pancreatic duct cells: a basis for defective HCO-3 secretion in CF.

Pancreatic dysfunction in patients with cystic fibrosis (CF) is felt to result primarily from impairment of ductal HCO-3 secretion. We provide molecular evidence for the expression of NBC-1, an electrogenic Na+-HCO-3 cotransporter (NBC) in cultured human pancreatic duct cells exhibiting physiological features prototypical of CF duct fragments (CFPAC-1 cells) or normal duct fragments [CAPAN-1 cells and CFPAC-1 cells transfected with wild-type CF transmembrane conductance regulator (CFTR)]. We further demonstrate that 1) HCO-3 uptake across the basolateral membranes of pancreatic duct cells is mediated via NBC and 2) cAMP potentiates NBC activity through activation of CFTR-mediated Cl- secretion. We propose that the defect in agonist-stimulated ductal HCO-3 secretion in patients with CF is predominantly due to decreased NBC-driven HCO-3 entry at the basolateral membrane, secondary to the lack of sufficient electrogenic driving force in the absence of functional CFTR.

Cloning, renal distribution, and regulation of the rat Na+-HCO3- cotransporter.

We recently reported the cloning and expression of a human kidney Na+-HCO3- cotransporter (NBC-1) (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111-19114, 1997). To expedite in vivo experimentation, we now report the cDNA sequence of rat kidney NBC-1. In addition, we describe both the organ and nephron segment distributions and the regulation of NBC-1 mRNA under three models of pH stress: chloride-depletion alkalosis (CDA), metabolic acidosis, and bicarbonate loading. Rat NBC-1 cDNA encodes an open reading frame of 1,035 amino acids, with 96 and 87% identity to human and salamander NBC-1, respectively. Rat NBC-1 mRNA is expressed at high levels in kidney and brain, with lower levels in colon, stomach, and heart. None appears in liver. In the kidney, NBC-1 is expressed mainly in the proximal tubule, with traces found in medullary thick ascending limb and papilla. HCO3- loading decreased NBC-1 mRNA levels, which were unchanged either by metabolic acidosis or by CDA.

Colonic H+-K+-ATPase is induced and mediates increased HCO3- reabsorption in inner medullary collecting duct in potassium depletion.

BACKGROUND: Potassium depletion increases HCO3- reabsorption in outer medullary collecting duct (OMCD) by activation of colonic (c) H-K-ATPase (HKA). The purpose of the current experiments was to examine the role of the isoforms of HKA in HCO3- reabsorption by terminal inner medullary collecting duct (IMCD) cells in potassium depletion. METHODS: Sprague-Dawley rats were fed a potassium-free diet and studied after 8 to 10 days. mRNA expression of HKA isoforms in terminal portion of inner medulla was examined and correlated with HCO3- reabsorption in the terminal IMCD. RESULTS: Gastric (g) HKA mRNA decreased whereas colonic (c) HKA mRNA expression was heavily induced in terminal portion of inner medulla in potassium depleted rats. Net HCO3- flux (JtCO2) in terminal IMCD increased in potassium depletion (4.56 to 7.06 pmol/min/mm tubule length, P < 0.001). In normal rats, 1 mM ouabain in perfusate had no effect on JtCO2, whereas 10 microM Schering 28080 (SCH) decreased JtCO2 to 2.4 (P < 0.002). In KD rats, 1 mM ouabain decreased JtCO2 to 4.9 (P < 0.005) and 10 microM SCH decreased JtCO2 to 3.3 (P < 0.001). However, the inhibitory effects of SCH and ouabain on JtCO2 in potassium depleted animals were not additive. CONCLUSIONS: The data indicate that gHKA is suppressed whereas cHKA is induced in potassium depletion and mediates increased HCO3- reabsorption in terminal IMCD. The results further indicate that cHKA in vivo is sensitive to both SCH and ouabain.

Activation of H+-ATPase by hypotonicity: a novel regulatory mechanism for H+ secretion in IMCD cells.

The effect of hypotonicity on H+-ATPase activity was examined in cultured inner medullary collecting duct (mIMCD-3) cells. mIMCD-3 cells were grown to confluence, loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and assayed for H+-ATPase activity measured as the Na+- and K+-independent intracellular pH (pHi) recovery following an acid load. Exposure of mIMCD-3 cells to a hypotonic solution (150 mosmol/kgH2O) increased pHi recovery by approximately 350% (P < 0.0001). This effect was inhibited by diethylstilbestrol (an inhibitor of H+-ATPase) and was not dependent on external K+, indicating lack of involvement of H+-K+-ATPase. H+-ATPase activation was acute, independent of cell calcium, and was not secondary to Cl- channel activation. The magnitude of H+-ATPase upregulation was dependent on the osmolarity of the media, with maximum stimulation at 150 mosmol/kgH2O. H+-ATPase upregulation in hypotonicity was significantly blocked in the presence of staurosporine or calphostin C or in cells pretreated with phorbol 12-myristate 13-acetate (PMA), indicating involvement of protein kinase C. Hypotonicity inhibited the Na+/H+ exchanger activity in mIMCD-3 cells, indicating that its stimulatory effect is specific to H+-ATPase. In conclusion, a novel regulatory mechanism of H+-ATPase by hypotonicity is described. The increased H+-ATPase activity in hypotonicity may be responsible for increased HCO-3 reabsorption and maintained acid-base homeostasis in hyposmolar states.

Functional characterization of a cloned human kidney Na+:HCO3- cotransporter.

Functional properties of a cloned human kidney Na+:HCO3- cotransporter (NBC-1) were studied in cultured HEK-293 cells that were transiently transfected with NBC-1 cDNA. The Na+:HCO3- cotransporter activity was assayed as the Na+ and HCO3-dependent pHi recovery from intracellular acidosis with the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In acid-loaded cells and in the presence of amiloride (to block Na+/H+ exchange), switching to a Na+-containing solution (115 mM) resulted in rapid pHi recovery only in the presence of HCO3-. This recovery was completely abolished by 300 microM 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. Replacing the Na+ with Li+ (115 mM) caused significant HCO3--dependent, DIDS-sensitive pHi recovery from intracellular acidosis, with Li+ showing lower affinity than Na+. Potassium (K+) had no affinity for the Na+:HCO3- cotransporter. The Na+-dependent HCO3- cotransport was abolished in the presence of 0.2 mM harmaline. The Na+:HCO3- cotransporter could also function in Na+:OH- cotransport mode, although only at high external pH (7.8). Based on functional similarities with the mammalian kidney experiments, we propose that NBC-1 is the proximal tubule Na+:HCO3- cotransporter.

ANG II controls Na(+)-K+(NH4+)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb.

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na(+)-K+(NH4+)-2Cl- cotransport. ANG II at 10(-16) to 10(-12) M inhibited 30-50% (P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10(-12) M ANG II level cotransport activity; eventually, 10(-6) M ANG II stimulated 34% cotransport activity (P < 0.003). Inhibition by 10(-12) M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10(-12) M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10(-6) M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus < or = 10(-12) M ANG II inhibits via 20-HETE, whereas > or = 5 x 10(-11) M ANG II stimulates via PKC Na(+)-K+(NH4+)-2Cl- cotransport; all ANG II effects involve AT1 receptors and PLC activation.