Changes in the activity and expression of protein phosphatase-1 accompany the differential regulation of NHE3 before and after the onset of hypertension in spontaneously hypertensive rats.

AIM: The Na(+) /H(+) exchanger NHE3 activity decreases in the proximal tubule of spontaneously hypertensive rats (SHRs) as blood pressure increases, and this reduction is correlated with higher NHE3 phosphorylation levels at the PKA consensus site serine 552. This study tested the hypothesis that this lowered NHE3 activity is associated with an increase in PKA activity and expression, and/or a decrease in protein phosphatase-1 (PP1) activity and expression. METHODS: Proximal tubule NHE3 activity was measured as the rate of bicarbonate reabsorption by stationary microperfusion. NHE3 phosphorylation and protein expression were determined by immunoblotting. PKA and PP1 activities were determined using specific substrates under optimal enzymatic conditions. RESULTS: The PKA activator, 6-MB-cAMP, increased the phosphorylation levels of NHE3 at serine 552 in the renal cortex; this increase happens to a much greater extent in young pre-hypertensive SHRs (Y-SHRs) compared to adult SHRs with established hypertension (A-SHRs). Likewise, the inhibitory effect of 6-MB-cAMP on NHE3 transport activity was much more pronounced in the proximal tubules of Y-SHRs than in those of A-SHRs. Renal cortical activity of PKA was not significantly different between Y-SHRs and A-SHRs. On the other hand, Y-SHRs exhibited higher protein phosphatase 1 (PP1) activity, and their expression of the PP1 catalytic subunit PP1α in the renal cortex was also higher than in A-SHRs. CONCLUSION: Collectively, these results support the idea that the lower NHE3 transport activity and higher phosphorylation occurring after the development of hypertension in SHRs are due, at least in part, to reduced PP1-mediated dephosphorylation of NHE3 at serine 552.

Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments.

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(+)-ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 ± 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10), 10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 ± 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules.

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 muM and 10 muM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 muM concentrations. Comparing control and REN concentration of 1 muM, JHCO(3)(-), nmol cm(-2) s(-1)-1,76+/-0,11(control)x1,29+/-0,08(REN 10 muM); P<0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 muM (JHCO(3)(-), nmol cm(-2) s(-)1-0.80+/-0.07(control)x0.60+/-0.06(REN 1 muM); P<0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na(+)/H(+)exchanger and H(+)-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.

Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells.

Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions.

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na+/H+ exchanger in proximal S3 segment: role of cytosolic calcium.

The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na+/H+ exchanger and on the [Ca2+]i were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca2+]i and after 6-min pi there was a new increase of [Ca2+]i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca2+]i. RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca2+]i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca2+]i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca2+]i in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca2+]i (at 10(-6) M aldosterone plus RU 486).

Cl- and regulation of pH by MDCK-C11 cells

The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 æM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 æM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 æM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 æM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 æM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.

Cl- and regulation of pH by MDCK-C11 cells.

The interaction between H(+) extrusion via H(+)-ATPase and Cl(-) conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH(4)Cl acid pulse was 0.136 +/- 0.008 pH units/min (dpHi/dt) in Na(+) Ringer and 0.032 +/- 0.003 in the absence of Na(+) (0 Na(+)). With 0 Na(+) plus the Cl(-) channel inhibitor NPPB (10 microM), recovery was reduced to 0.014 +/- 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl(-) channels, increased dpHi/dt in 0 Na(+) to 0.061 +/- 0.009 and also in the presence of 46 nM concanamycin and 50 microM Schering 28080. Since it is thought that the Cl(-) dependence of H(+)-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 microM valinomycin at high (100 mM) K(+) was tested in our cells. In Na(+) Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl(-) channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 microM ATP was studied in 0 Na(+) Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 microM Bapta. We have shown that H(+)-ATPase present in MDCK C11 cells depends on Cl(-) ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H(+) transport.

Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet.

Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule.

Effect of chronic fish oil supplementation on renal function of normal and cachectic rats

In the present study we determined the effect of chronic diet supplementation with n-3 PUFA on renal function of healthy and cachectic subjects by providing fish oil (1 g/kg body weight) to female rats throughout pregnancy and lactation and then to their offspring post-weaning and examined its effect on renal function parameters during their adulthood. The animals were divided into four groups of 5-10 rats in each group: control, control supplemented with fish oil (P), cachectic Walker 256 tumor-bearing (W), and W supplemented with fish oil (WP). Food intake was significantly lower in the W group compared to control (12.66 ± 4.24 vs 25.30 ± 1.07 g/day). Treatment with fish oil significantly reversed this reduction (22.70 ± 2.94 g/day). Tumor growth rate was markedly reduced in the P group (16.41 ± 2.09 for WP vs 24.06 ± 2.64 g for W). WP group showed a significant increase in mean glomerular filtration rate compared to P and control (1.520 ± 0.214 ml min-1 kg body weight-1; P < 0.05). Tumor-bearing groups had low urine osmolality compared to control rats. The fractional sodium excretion decreased in the W group compared to control (0.43 ± 0.16 vs 2.99 ± 0.87 percent; P < 0.05), and partially recovered in the WP group (0.90 ± 0.20 percent). In summary, the chronic supplementation with fish oil used in this study increased the amount of fat in the diet by only 0.1 percent, but caused remarkable changes in tumor growth rate and cachexia, also showing a renoprotective function.

Effect of chronic fish oil supplementation on renal function of normal and cachectic rats.

In the present study we determined the effect of chronic diet supplementation with n-3 PUFA on renal function of healthy and cachectic subjects by providing fish oil (1 g/kg body weight) to female rats throughout pregnancy and lactation and then to their offspring post-weaning and examined its effect on renal function parameters during their adulthood. The animals were divided into four groups of 5-10 rats in each group: control, control supplemented with fish oil (P), cachectic Walker 256 tumor-bearing (W), and W supplemented with fish oil (WP). Food intake was significantly lower in the W group compared to control (12.66 +/- 4.24 vs 25.30 +/- 1.07 g/day). Treatment with fish oil significantly reversed this reduction (22.70 +/- 2.94 g/day). Tumor growth rate was markedly reduced in the P group (16.41 +/- 2.09 for WP vs 24.06 +/- 2.64 g for W). WP group showed a significant increase in mean glomerular filtration rate compared to P and control (1.520 +/- 0.214 ml min-1 kg body weight-1; P < 0.05). Tumor-bearing groups had low urine osmolality compared to control rats. The fractional sodium excretion decreased in the W group compared to control (0.43 +/- 0.16 vs 2.99 +/- 0.87%; P < 0.05), and partially recovered in the WP group (0.90 +/- 0.20%). In summary, the chronic supplementation with fish oil used in this study increased the amount of fat in the diet by only 0.1%, but caused remarkable changes in tumor growth rate and cachexia, also showing a renoprotective function.

Role of luminal anion and pH in distal tubule potassium secretion.

Potassium secretory flux (J(K)) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, J(K) was measured with a double-barreled K(+) electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl(-) had been replaced with a less permeant anion and/or pH had been increased to 8.0. J(K) increased when Cl(-) was replaced by either acetate ( approximately 37%), sulfate ( approximately 32%), or bicarbonate ( approximately 62%), and also when the pH of the control perfusate was increased ( approximately 26%). The majority (80%) of acetate-stimulated J(K) was Ba(2+) sensitive, but furosemide (1 mM) further reduced secretion ( approximately 10% of total), suggesting that K(+)-Cl(-) cotransport was operative. Progressive reduction in luminal Cl(-) concentration from 100 to 20 to 2 mM caused increments in J(K) that were abolished by inhibitors of K(+)-Cl(-) cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased J(K) even in the presence of Ba(2+), suggesting that this effect cannot be accounted for only by K(+) channel modulation of K(+) secretion in the distal nephron of the rat. Collectively, these data suggest a role for K(+)-Cl(-) cotransport in distal nephron K(+) secretion.

Factors affecting ammonium uptake by C11 clone of MDCK cells.

In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.

Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187.

The regulated Cl(-) secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca(2+), cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux (J(w) = -0.16 +/- 0.02 microl.min(-1).cm(-2)) in parallel with moderate short-circuit current values (I(sc) = 1.55 +/- 0.23 microA/cm(2)). The secretory J(w) reversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I(sc), but, unexpectedly, J(w) was not affected. Bumetanide, an inhibitor of basolateral Na(+)-K(+)-2Cl(-) cotransporter, completely blocked secretory J(w) with no change in I(sc). Conversely, serosal forskolin increased I(sc), but J(w) switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J(w) and I(sc). No difference between the absorptive and secretory unidirectional Cl(-) fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl(-) flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J(w) values cannot be shown by I(sc) and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.

Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-).

The role of H(+)-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused "in vitro" while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na(+)- and Cl(-)-free medium, cell pH fell by a mean of 0.37+/-0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 +/-0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl(-) channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na(+) solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018+/- 0.0021 min(-1), not significantly different from zero (P>0.42). For superfusion with Na(+)0 Ringer, this variation was 0.081+/-0.015 min(-1), P<0.001 against 0. These slopes were markedly reduced by the Cl(-) channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na(+)-free medium is probably due to a vacuolar H(+)-ATPase, whose activity is stimulated in the presence of Cl(-), and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl(-) and on the integrity of the cytoskeleton.

Control of cell pH in the T84 colon cell line.

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 +/- 0.023 (n = 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 +/- 0.011 pH units/min in the presence of Na+, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO3- Ringer, basal cell pH was 7.21 +/- 0.020 (n = 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of -0.14 +/- 0.012 (n = 8) pH/min; this return was retarded or abolished in the absence of Cl- or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl-/HCO3- exchange. This exchange was not Na+ dependent. When Na+ was added to cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, cell alkalinization by 0.19 +/- 0.04 (n = 11) pH units was observed, suggesting the presence of Na+/HCO3- cotransport carrying HCO3- into these cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl-/HCO3- exchange and Na+/HCO3- cotransport participate in cell pH regulation in T84 cells.

Chronic effect of parathyroid hormone on NHE3 expression in rat renal proximal tubules.

BACKGROUND: The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. METHODS: Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. RESULTS: Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HP rats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HP rats and increased 54.6 +/- 7.8% in PTX rats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HP rats and was increased in PTX rats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HP rats and 27. 7 +/- 2.1% increased in PTX. CONCLUSIONS: Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance.

V(1) receptors in luminal action of vasopressin on distal K(+) secretion.

Luminal perfusion with collected proximal fluid increases distal K(+) secretion compared with artificial solutions. Arginine vasopressin (AVP), present in luminal fluid, might be responsible for this observation. K(+) secretion rate (J(K)) was measured by K(+)-sensitive microelectrodes during paired luminal stationary microperfusion with control and AVP-containing 0.5 mM K(+) solutions. J(K) was 1.34 +/- 0.35 (n = 24 tubules) nmol x cm(-2) x s(-1) during perfusion with 10(-9) M AVP, against 0.90+/-0.12 nmol x cm(-2) x s(-1) (n = 21) in control (P<0.02). With 10(-9) M AVP+10(-6) M beta-mercapto-beta-beta-cyclopenta-methylenepropionyl(1), O-Me-Tyr(2)-Arg(8) vasopressin (MCMV), a specific peptide V(1)-receptor antagonist, J(K) was 0.36+/-0.067 against 0.77+/-0.10 (control; n = 9) nmol x cm(-2) x s(-1) (P<0.01). With 10(-6) M MCMV alone, J(K) was 0.37+/-0.04 against a control of 0.62+/-0.06 (n = 19) nmol. cm(-2). s(-1) (P<0.01). A peptide V(2) antagonist had no such effect. In Brattleboro rats, which do not produce endogenous AVP, MCMV had no effect when given alone, although AVP still stimulated J(K). In conclusion, luminal AVP stimulates distal J(K) significantly. The V(1) antagonist MCMV inhibits the effect of AVP but also reduces J(K) when given alone. This suggests that AVP acts luminally via V(1) receptors but also that there appears to be a background effect of endogenous AVP blocked by the antagonist.