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ABSTRACT: Neurotrophins are expressed in the adult kidney, but their significance is unclear. We showed previously that nerve growth factor (NGF) inhibits HCO absorption in the rat medullary thick ascending limb (MTAL) via an extracellular signal-regulated kinase (ERK)-dependent pathway. Here we examined whether other neurotrophic factors affect MTAL HCO absorption. Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor had no effect. In contrast, neurotrophin-3 (NT-3, 0.7 nM) inhibited HCO absorption by 40% (half-maximal inhibition at approximately 0.4 nM). Inhibition by NT-3 was additive to inhibition by NGF. Inhibitors of ERK activation that block inhibition by NGF had no effect on inhibition by NT-3. In contrast, 8-bromo-cAMP or forskolin pretreatment blocked inhibition by NT-3 but not NGF. Inhibition by NT-3 was also blocked by the specific protein kinase A (PKA) inhibitor myristoylated PKI(14-22) amide and by vasopressin, which inhibits HCO absorption via cAMP. Inhibitors of phosphatidylinositol 3-kinase or protein kinase C did not affect NT-3-induced inhibition, but inhibition by NT-3 was eliminated by genistein, consistent with involvement of a receptor tyrosine kinase. These results demonstrate that NT-3 inhibits HCO absorption via a cAMP- and PKA-dependent pathway. NT-3 and NGF regulate MTAL ion transport through different signal transduction mechanisms. These studies establish a direct role for NT-3 in regulation of renal tubule transport and identify the MTAL as an important target for neurotrophins, which may be involved in the control of renal acid excretion.
AJP Cell Physiology 01/2002; 281(6):C1804-11. · 3.54 Impact Factor
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ABSTRACT: The signal transduction mechanisms that mediate osmotic regulation of Na(+)/H(+) exchange are not understood. Recently we demonstrated that hyposmolality increases HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through stimulation of the apical membrane Na(+)/H(+) exchanger NHE3. To investigate the mechanism of this stimulation, MTALs from rats were isolated and perfused in vitro with 25 mM HCO(3)(-)-containing solutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (100 nM) and LY-294002 (20 microM) blocked completely the stimulation of HCO(3)(-) absorption by hyposmolality. In tissue strips dissected from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs, hyposmolality increased PI 3-K activity 2. 2-fold. Wortmannin blocked the hyposmolality-induced PI 3-K activation. Further studies examined the interaction between hyposmolality and vasopressin, which inhibits HCO(3)(-) absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO(3)(-) absorption, due to an effect of cAMP to inhibit hyposmolality- induced activation of PI 3-K. In contrast to their effects to block stimulation by hyposmolality, PI 3-K inhibitors and vasopressin have no effect on inhibition of apical Na(+)/H(+) exchange (NHE3) and HCO(3)(-) absorption by hyperosmolality. These results indicate that hyposmolality increases NHE3 activity and HCO(3)(-) absorption in the MTAL through activation of a PI 3-K-dependent pathway that is inhibited by vasopressin and cAMP. Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediated through different signal transduction mechanisms.
AJP Cell Physiology 12/2000; 279(5):C1443-54. · 3.54 Impact Factor
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ABSTRACT: The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.
Journal of Clinical Investigation 01/2000; 104(11):1593-602. · 15.39 Impact Factor
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ABSTRACT: The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10(-8) M in the bath) inhibits HCO-3 absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 microM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 microM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M), the major P-450 metabolite in the MTAL, inhibited HCO-3 absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10(-8) M) to the bath had no effect on HCO-3 absorption. The inhibition of HCO-3 absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 microM) or herbimycin A (1 microM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10 microM) or the NO synthase (NOS) substrate L-arginine (1 mM) to the bath stimulated HCO-3 absorption by 35%, suggesting that NO directly regulates MTAL HCO-3 absorption. Addition of 10(-11) to 10(-10) M ANG II to the bath did not affect HCO-3 absorption. We conclude that ANG II inhibits HCO-3 absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of HCO-3 absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.
The American journal of physiology 05/1999; 276(5 Pt 2):F726-36.
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ABSTRACT: Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.
Journal of Biological Chemistry 04/1999; 274(12):7841-7. · 4.77 Impact Factor
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ABSTRACT: Mitogen-activated protein (MAP) kinases are activated by osmotic stress in a variety of cells, but their function and regulation in renal tubules is poorly understood. The present study was designed to examine the osmotic regulation of MAP kinases in the medullary thick ascending limb (MTAL) of the rat and to determine their possible role in the hyperosmotic inhibition of HCO-3 absorption in this segment. Tissues from the inner stripe of the outer medulla and microdissected MTALs were incubated at 37 degreesC in control (290 mosmol/kgH2O) or hyperosmotic (300 mM added mannitol) solution for 15 min. Activities of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase were then measured using immune complex assays. Hyperosmolality increased p38 MAP kinase activity (2.3-fold) and ERK activity (2.0-fold) but had no effect on JNK activity (1.1-fold). Exposure to hyperosmolality for various times showed that the activation of p38 MAP kinase was rapid (</=5 min) and was sustained for up to 60 min, whereas the activation of ERK was transient (ERK activity peaked at 15 min, then declined to basal levels at 30 min). Pretreatment with the MAP kinase kinase inhibitor PD98059 (15 microM) blocked the hyperosmotic activation of p38 MAP kinase and ERK but did not prevent hyperosmotic inhibition of HCO-3 absorption. These results show that hyperosmolality differentially activates p38 MAP kinase and ERK in the MTAL. In contrast, we found no evidence for involvement of JNK in the early response to hyperosmotic stress. Eliminating the activation of p38 MAP kinase and ERK does not prevent hyperosmotic inhibition of HCO-3 absorption, suggesting that hyperosmolality inhibits apical membrane Na+/H+ exchange (NHE3) activity via a signaling pathway distinct from these MAP kinase pathways.
The American journal of physiology 11/1998; 275(4 Pt 2):F478-86.
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D W Good
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ABSTRACT: Growth factors stimulate Na+/H+ exchange activity in many cell types but their effects on acid secretion via this mechanism in renal tubules are poorly understood. We examined the regulation of HCO3- absorption by nerve growth factor (NGF) in the rat medullary thick ascending limb (MTAL), which absorbs HCO3- via apical membrane Na+/H+ exchange. MTAL were perfused in vitro with 25 mM HCO3- solutions (pH 7.4; 290 mosmol/kgH2O). Addition of 0.7 nM NGF to the bath decreased HCO3- absorption from 13.1 +/- 1.1 to 9.6 +/- 0.8 pmol.min-1.mm-1 (P < 0.001). In contrast, with 10(-10) M arginine vasopressin (AVP) in the bath, addition of NGF to the bath increased HCO3- absorption from 8.0 +/- 1.6 to 12.5 +/- 1.3 pmol.min-1.mm-1 (P < 0.01). Both effects of NGF were blocked by genistein, consistent with the involvement of tyrosine kinase pathways. However, the AVP-dependent stimulation required activation of protein kinase C (PKC), whereas the inhibition was PKC independent, indicating that the NGF-induced signaling pathways leading to inhibition and stimulation of HCO3- absorption are distinct. Hypertonicity blocked the inhibition but not the AVP-dependent stimulation, suggesting that hypertonicity and NGF may inhibit HCO3- absorption via a common mechanism. These data demonstrate that NGF inhibits HCO3- absorption in the MTAL under basal conditions but stimulates HCO3- absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is a critical determinant of the response of the MTAL to growth factor stimulation and suggest that NGF can either inhibit or stimulate apical Na+/H+ exchange activity depending on its interactions with other regulatory factors. Locally produced growth factors such as NGF may play a role in regulating renal tubule HCO3- absorption.
The American journal of physiology 04/1998; 274(4 Pt 1):C931-9.
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ABSTRACT: The effects of long-term exposure to hyperosmotic medium on the Na+/H+ exchanger isoform NHE-3 were examined in cultured renal epithelial cells (LLC-PK1). LLC-PK1 cells were grown to confluence in control medium (310 mOsm/kg H2O) and then either switched to a hyperosmotic medium (510 mOsm/kg H2O; addition of NaCl or mannitol) or maintained in the control medium for 48 hours. The Na+/H+ exchanger activity was then assessed in isosmotic solutions by measurement of amiloride-sensitive acid-stimulated 22Na+ influx or Na+-dependent acid extrusion. Acid-stimulated 22Na+ influx was decreased significantly in cells incubated in hyperosmotic medium (10.5 +/- 0.9 nmol/mg protein, control vs. 5.8 +/- 0.6, hyperosmotic; P < 0.01). Incubation in hyperosmotic medium also decreased the initial rate of Na+-dependent acid extrusion by approximately 60% over the intracellular pH range 6.9 to 7.3. Intracellular buffering power did not differ in the control and hyperosmotic groups. The Na+/H+ exchanger isoform NHE-3 mRNA and protein, assessed by Northern hybridization and immunoblot analysis, respectively, were unchanged in LLC-PK1 cells incubated in hyperosmotic medium compared with controls, suggesting post-translational regulation by high osmolality. These results demonstrate that long-term exposure to hyperosmotic medium causes an adaptive decrease in Na+/H+ exchange (NHE-3) activity in LLC-PK1 cells, and that this effect is unlikely to involve antiporter gene regulation or a change in protein abundance.
Kidney International 02/1998; 53(2):423-31. · 6.61 Impact Factor
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ABSTRACT: In the medullary thick ascending limb (MTAL) of the rat kidney, prostaglandin E2 (PGE2) reverses inhibition of HCO3 absorption by arginine vasopressin (AVP). This effect of PGE2 is blocked by chelerythrine or staurosporine and mimicked by phorbol ester, suggesting a critical role for protein kinase C (PKC). The present study was designed to examine directly regulation of PKC isoforms by PGE2 in the inner stripe of the outer medulla and in microdissected MTALs. Immunoblots with isoform-specific anti-PKC antibodies detected alpha-, beta II-, delta-, epsilon-, and zeta-isoforms in both inner stripe and MTAL. The beta I- and gamma-isoforms were not detected. Translocation and activation of PKC were assessed by immunoblot analysis and by direct measurement of enzyme activity using an immune complex kinase assay. In inner stripe tissue incubated with 10(10) M AVP, PGE2 10(6) M for 20 min) induced translocation of PKC-delta from the cytosolic fraction to the membrane fraction. This translocation was associated with an 85% increase in PKC-delta activity in the membrane fraction and a 70% decrease in PKC-delta activity in the cytosolic fraction. PGE2 had no effect on the subcellular distribution or the activities of the other isoforms. Activation of PKC-delta was confirmed directly in microdissected MTALs, in which PGF2 caused a near complete loss of PKC-delta from the cytosolic fraction. PGE2 did not induce translocation of PKC-delta in the absence of AVP. These results demonstrate that 1) the MTAL expresses Ca(2+)-dependent (alpha, beta II) and Ca(2+)-independent (delta, epsilon, zeta) PKC isoforms; 2) PGE2 causes selective activation of PKC-delta, which likely mediates the action of PGE2 to reverse AVP inhibition of HCO-3 absorption; and 3) PGE2 activation of PKC-delta requires the presence of AVP, which may explain the fact that PGE2 influences HCO-3 transport only when AVP is present.
The American journal of physiology 06/1997; 272(5 Pt 2):F624-31.
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ABSTRACT: Arginine vasopressin (AVP) inhibits HCO3- absorption (JHCO3) in the medullary thick ascending limb (MTAL) of the rat by increasing adenosine 3', 5'-cyclic monophosphate. Hyperosmolality also inhibits JHCO3 via a pathway additive to inhibition by AVP. To determine whether these regulatory effects are modulated by prostaglandin E2 (PGE2), MTAL were isolated and perfused in vitro with 25 mM HCO3- solutions (pH 7.4; 290 mosmol/kgH2O). PGE2 (10(-6) M in the bath) had no effect on JHCO3 in the absence of AVP. In contrast, with 10(-10) MAVP in the bath solution, addition of 10(-8) or 10(-6) M PGE2 to the bath increased JHCO3 from 9.7 +/- 0.8 to 14.3 +/- 1.1 pmol.min-1.mm-1 (P < 0.001). In the presence of AVP and hyperosmolality (75 mM NaCl added to perfusate and bath), PGE2 increased JHCO3 from 1.4 +/- 0.1 to 7.5 +/- 0.5 pmol.min-1.mm-1 (P < 0.005). PGE2 also stimulated JHCO3 in the presence of AVP and hypertonic urea. Cholera toxin (CTX, 10(-12)-10(-9) M in the bath) inhibited JHCO3 by 40%, and this inhibition was reversed by PGE2. PGE2 did not reverse inhibition of JHCO3 by forskolin. The stimulation of JHCO3 by PGE2 in the presence of AVP was blocked by pretreatment with pertusis toxin (PTX, 2 x 10(-11) or 10(-8) M). Neither CTX nor PTX affected inhibition of JHCO3 by hyperosmolality. These results demonstrate that PGE2 reverses inhibition of JHCO3 by AVP by acting via a PTX-sensitive G protein (presumably Gi) to inhibit AVP-stimulated adenosine 3', 5'-cyclic monophosphate production. PGE2 may act as a counterregulatory factor to maintain a stable rate of HCO3- absorption in the MTAL during antidiuresis when circulating AVP levels and medullary osmolality are elevated.
The American journal of physiology 06/1996; 270(5 Pt 2):F711-7.
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D W Good
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ABSTRACT: In the medullary thick ascending limb (MTAL) of the rat, prostaglandin E2 (PGE2) reverses inhibition of HCO3- absorption (JHCO3) by arginine vasopressin (AVP) by inhibiting AVP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production. To determine whether this regulation by PGE2 involves protein kinase C (PKC), MTAL segments were perfused in vitro with physiological solutions containing 25 mM HCO3- (pH 7.4). With 10(-10) MAVP in the bath, addition of 10(-6) M PGE2 to the bath increased JHCO3 from 7.8 +/- 0.4 to 13.0 +/- 1.1 pmol.min-1.mm-1 (P < 0.01). This effect was blocked completely by pretreatment with the PKC inhibitors staurosporine or chelerythrine chloride (10(-7) M in the bath). With both AVP and PGE2 in the bath, addition of staurosporine or chelerythrine to the bath decreased JHCO3 from 12.2 +/- 1.1 to 7.3 +/- 0.6 pmol.min-1.mm-1 (P < 0.005). Neither staurosporine nor chelerythrine affected JHCO3 under basal conditions or in the presence of AVP alone. With AVP in the bath, addition of phorbol 12-myristate 13-acetate (PMA, 10(-6) M) to the bath increased JHCO3 from 5.0 +/- 0.5 to 9.1 +/- 1.0 pmol.min-1.mm-1 (P < 0.01). Similar to PGE2, PMA had no effect on JHCO3 in the absence of AVP or in the presence of 10(-6) M bath forskolin. The effect of PMA to stimulate JHCO3 in the presence of AVP was abolished by pretreatment with pertussis toxin (2 x 10(-11) M). We conclude that 1) PGE2 reverses AVP inhibition of HCO3- absorption by activation of PKC, 2) PKC likely increases JHCO3 by inhibiting AVP-stimulated cAMP production via a Gi-dependent mechanism, and 3) PKC activity has no influence on basal HCO3- absorption rate.
The American journal of physiology 06/1996; 270(6 Pt 2):F978-85.
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ABSTRACT: The medullary thick ascending limb (MTAL) of the rat actively absorbs both HCO3- and ammonium. The roles of apical membranes Na+/H+ exchange in these processes and in determining steady-state intracellular pH (pHi) were examined in MTAL perfused in vitro with solutions containing 146 mM Na+ and 25 mM HCO3- (pH 7.4). Addition of 1 mM amiloride or 50 microM ethylisopropylamiloride (EIPA) to the lumen decreased HCO3- absorption (JHCO3) from 10.6 +/- 0.5 to 2.3 +/- 0.3 pmol.min-1.mm-1 (P < 0.001) and pHi from 7.10 +/- 0.02 to 6.86 +/- 0.03 (P < 0.001). The combination of lumen Na+ replacement plus amiloride abolished JHCO3. Chronic metabolic acidosis (CMA) caused a 32% increase in JHCO3 that was inhibited by luminal amiloride. Addition of 4 mM NH4Cl to perfusate and bath markedly decreased pHi (from 7.10 to 6.70) but did not stimulate luminal H+ secretion as assessed by HCO3- absorption. With 4 mM NH4Cl in perfusate and bath, luminal addition of amiloride decreased pHi from 6.70 +/- 0.06 to 6.50 +/- 0.05 (P < 0.005) but had no effect on net ammonium absorption. These results demonstrate that 1) apical membrane Na+/H+ exchange mediates virtually all of HCO3- absorption and is an important determinant of steady-state pHi in the MTAL; 2) the adaptive increase in HCO3- absorption in CMA is mediated by an increase in apical membrane Na+/H+ exchange; 3) ammonium markedly acidifies the cells but does not stimulate luminal acidification, suggesting that pHi is not a predominant influence on apical Na+/H+ exchange activity and that H+ generated in the cells as the result of transcellular ammonium absorption is extruded across the basolateral membrane; and 4) apical membrane Na+/H+ exchange is not important for ammonium absorption.
The American journal of physiology 04/1996; 270(4 Pt 2):F691-9.
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ABSTRACT: The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity.
Proceedings of the National Academy of Sciences 01/1996; 92(26):12525-9. · 9.68 Impact Factor
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ABSTRACT: The role of renal ammonium excretion in the maintenance of chronic metabolic alkalosis is poorly defined, particularly under conditions in which the alkalosis is associated with secondary potassium depletion. Therefore, free-flow micropuncture experiments were performed to examine the effects of chronic chloride depletion metabolic alkalosis (CDAlk) on renal ammonium production, urinary ammonium excretion, and proximal convoluted tubule (PCT) ammonium transport in the rat in vivo. CDAlk was generated by peritoneal dialysis against NaHCO3 and maintained for 6-7 days by dietary Cl- restriction. Pair-fed controls were dialyzed against NaCl. Rats with CDAlk had elevated plasma HCO3- concentration, hypokalemia, and hypochloremia. HCO3- excretion was negligible in both control and CDAlk rats. Glomerular filtration rate and urine pH did not differ. CDAlk reduced urinary ammonium excretion by 35% but had no significant effect on whole kidney ammonium production. Net secretion of ammonium by the PCT was decreased by 70% and absolute delivery of ammonium out of the PCT was decreased by 55% in the CDAlk rats. The decrease in PCT ammonium secretion was the combined result of a decrease in net ammonium secretion along the early PCT and an increase in net ammonium absorption along the late PCT.(ABSTRACT TRUNCATED AT 250 WORDS)
The American journal of physiology 11/1995; 269(4 Pt 2):F508-14.
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ABSTRACT: Regulation of HCO3- and Cl- absorption by arginine vasopressin (AVP) and prostaglandin E2 (PGE2) was examined in isolated, perfused medullary thick ascending limbs (MTAL) from 4- to 7-wk-old spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. AVP inhibited HCO3- absorption by 50% at 10(-10) M and by 25% at 2 x 10(-12) M in MTAL from both WKY and SHR. Cholera toxin (10(-9) M) or forskolin (10(-6) M) in the bath also inhibited HCO3- absorption by 50% in the SHR. In MTAL from WKY, PGE2 (10(-6) M in the bath) increased HCO3- absorption from 7.1 +/- 0.4 to 12.0 +/- 0.4 pmol.min-1.mm-1 (P < 0.005) and decreased Cl- absorption from 65 +/- 7 to 47 +/- 6 pmol.min-1.mm-1 (P < 0.001) in the presence of 10(-10) M AVP. Under the same conditions, PGE2 had no effect on HCO3- or Cl- absorption in MTAL from SHR. PGE2 also reversed submaximal inhibition of HCO3- absorption by 2 x 10(-12) M AVP in WKY but not in SHR. With 10(-10) M AVP in the bath, phorbol 12-myristate 13-acetate (10(-6) M in the bath) increased HCO3- absorption from 6.6 +/- 0.5 to 12.3 +/- 0.4 pmol.min-1.mm-1 in MTAL from WKY and from 7.6 +/- 0.7 to 12.6 +/- 1.2 pmol.min-1.mm-1 in MTAL from SHR (P < 0.005). These results demonstrate that 1) the effects of PGE2 to stimulate HCO3- absorption and inhibit Cl- absorption in the presence of AVP are absent in MTAL from SHR, 2) the defect may involve an inability of PGE2 to stimulate protein kinase C, and 3) regulation of HCO3- absorption by AVP via adenosine 3',5'-cyclic monophosphate is similar in MTAL from WKY and SHR. The lack of PGE2 inhibition of NaCl absorption in the MTAL may contribute to renal salt retention during the development of hypertension in the SHR.
The American journal of physiology 08/1995; 269(1 Pt 2):F47-54.
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D W Good
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ABSTRACT: In the rat medullary thick ascending limb (MTAL), hyperosmolality inhibits transepithelial HCO3- absorption (JHCO3-) by inhibiting apical membrane Na+/H+ exchange. To examine signaling mechanisms involved in this regulatory response, MTALs were isolated and perfused in vitro with 25 mM HCO3- solutions (290 mosmol/kg H2O). Osmolality was increased in lumen and bath solutions by addition of 300 mM mannitol or 75 mM NaCl. Addition of mannitol reduced JHCO3- by 60% and addition of NaCl reduced JHCO3- by 50%. With the protein tyrosine kinase (PTK) inhibitor genistein (7 microM) or herbimycin A (1 microM) in the bath, addition of mannitol reduced JHCO3- only by 11% and addition of NaCl reduced JHCO3- only by 15%. Staurosporine (10(-7) M) or forskolin (10(-6) M) in the bath had no effect on inhibition of JHCO3- by hypertonic NaCl. Genistein had no effect on inhibition of JHCO3- by vasopressin (a cyclic AMP-dependent process) or stimulation of JHCO3- by prostaglandin E2 (a protein kinase C-dependent process). Under isosmotic conditions, addition of genistein or herbimycin A to the bath increased JHCO3- by 30% through stimulation of apical membrane Na+/H+ exchange. Addition of the tyrosine phosphatase inhibitor molybdate (50 microM) to the bath reproduced the inhibition of JHCO3- observed with hyperosmolality. These data indicate that 1) the effect of hyperosmolality to inhibit MTAL HCO3- absorption through inhibition of apical membrane Na+/H+ exchange is mediated via a PTK-dependent pathway that functions independent of regulation by cyclic AMP and protein kinase C, and 2) a constitutive PTK activity inhibits apical membrane Na+/H+ exchange and HCO3- absorption under isosmotic conditions. Our results suggest that tyrosine phosphorylation is a critical step in inhibition of the apical Na+/H+ exchanger isoform NHE-3 by hyperosmolality.
Journal of Biological Chemistry 05/1995; 270(17):9883-9. · 4.77 Impact Factor
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ABSTRACT: Apical membrane Na+/H+ exchange mediates virtually all of transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). Regulation of the apical exchanger by intracellular pH (pHi) and hyperosmolality was studied in the isolated, perfused MTAL by measurement of pHi using the fluorescent probe 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein. Under isosmotic conditions (290 mosmol/kg H2O), the Na+/H+ exchange rate increased sigmoidally over the pHi range 7.8 to 6.5 (Hill coefficient = 2.1), consistent with cooperative activation of the exchanger by internal H+. The exchanger had a high apparent affinity for intracellular H+ (apparent pK = 7.36), which resulted in the exchanger being maximally active at resting pHi and insensitive to changes in pHi over the physiologic pHi range (6.5-7.2). Hyperosmolality (590 mosmol/kg H2O) inhibited Na+/H+ exchange by at least 35% at all pHi values studied and induced pHi dependence of the exchanger between 6.5 and 7.2. The inhibition by hyperosmolality appeared to be the result of an acid shift of the pHi dependence curve of the exchanger. These functional properties of apical membrane Na+/H+ exchange can account for our previous observations that hyperosmolality inhibited net HCO3- absorption and that the rate of HCO3- absorption did not correlate with pHi. Apical membrane Na+/H+ exchange in the MTAL differs functionally from Na+/H+ exchange in other cell types in which exchanger activity is stimulated rather than inhibited by hyperosmolality.
Journal of Biological Chemistry 09/1994; 269(32):20250-5. · 4.77 Impact Factor
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ABSTRACT: The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium-sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)-NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.
The Journal of General Physiology 06/1994; 103(5):917-36. · 3.84 Impact Factor
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D W Good
Annual Review of Physiology 02/1994; 56:623-47. · 20.83 Impact Factor
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D W Good
Seminars in Nephrology 04/1993; 13(2):225-35. · 2.12 Impact Factor
