Search PubMed⌕ Search

Biomedical subjects

O W Moe

Publications and source records attributed to O W Moe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1).

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.

Adenosine↗
PubMed1999

Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase a and NHE-3 phosphoserines 552 and 605.

Regulation of the renal Na/H exchanger NHE-3 by protein kinase A (PKA) is a key intermediate step in the hormonal regulation of acid-base and salt balance. We studied the role of NHE-3 phosphorylation in this process in NHE-deficient AP-1 cells transfected with NHE-3 and in OKP cells expressing native NHE-3. A dominant-negative PKA-regulatory subunit completely abolished the effect of cAMP on NHE-3 activity demonstrating a role of PKA in the functional regulation of NHE-3 by cAMP. NHE-3 isolated from cAMP-treated cells showed lower phosphorylation by purified PKA in vitro suggesting that NHE-3 is a PKA substrate in vivo. Although changes in NHE-3 whole protein phosphorylation is difficult to detect in response to cAMP addition, the tryptic phosphopeptide map of in vivo phosphorylated NHE-3 showed a complex pattern of constitutive and cAMP-induced phosphopeptides. To test the causal relationship between phosphorylation and activity, we mutated eight serines in the cytoplasmic domain to glycine or alanine. Single or multiple mutants harboring S552A or S605G showed no PKA activation or reduced regulation by PKA activation. Ser-552 and Ser-605 were phosphorylated in vivo. However, multiple mutations of serines other than Ser-552 or Ser-605 also reduced the functional PKA regulation. We conclude that regulation of NHE-3 by PKA in vivo involves complex mechanisms, which include phosphorylation of Ser-552 and Ser-605.

Amino Acid Substitution↗
PubMed1999

Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation.

Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3'-triiodothyronine (T3) increases NHE3 activity. T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T3 stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5'-flanking region of the rat NHE3 gene was stimulated by T3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance.

Animals↗
PubMed1999

ETB receptor activation leads to activation and phosphorylation of NHE3.

In OKP cells expressing ETB endothelin receptors, activation of Na+/H+ antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na+/H+ exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in cells expressing ETB receptors but not in cells expressing ETA receptors. Receptor specificity was not due to demonstrable differences in receptor-specific activation of tyrosine phosphorylation pathways or inhibition of adenylyl cyclase. Phosphorylation was associated with a decrease in mobility on SDS-PAGE, which was reversed by treating immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was first seen at 5 min and was maximal at 15-30 min. Phosphorylation was maximal with 10(-9) M ET-1. Phosphorylation occurred on threonine and serine residues at multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP cells on multiple threonine and serine residues. ETB receptor specificity, time course, and concentration dependence are all similar between ET-1-induced increases in NHE3 activity and phosphorylation, suggesting that phosphorylation plays a key role in activation.

Animals↗
PubMed1999

Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation.

Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.

Animals↗
PubMed1999

Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3).

In the absence of exogenous glucocorticoids, decreasing media pH (from 7.4 to 6.8) for 24 hours increased the Na+/H+ exchanger 3 (NHE3) activity in opossum kidney (OKP) cells. 10(-7) M and 10(-8) M hydrocortisone increased NHE3 activity, and in their presence, acid incubation further increased NHE3 activity. Hydrocortisone (10(-9) M) had no effect on NHE3 activity, but in its presence, the effect of acid incubation on NHE3 activity increased twofold. Aldosterone (10(-8) M) had no effect. In the absence of hydrocortisone, acid incubation increased NHE3 protein abundance by 47%; in the presence of 10(-9) M hydrocortisone, acid incubation increased NHE3 protein abundance by 132%. The increase in NHE3 protein abundance was dependent on protein synthesis. However, 10(-9) M hydrocortisone did not modify the effect of acid incubation to cause a twofold increase in NHE3 mRNA abundance. In the absence of protein synthesis, 10(-9) M hydrocortisone did potentiate an effect of acid on NHE3 activity, which was due to trafficking of NHE3 to the apical membrane. These results suggest that glucocorticoids and acid interact synergistically at the level of NHE3 translation and trafficking.

Animals↗
PubMed1998

Chronic hyperosmolality increases NHE3 activity in OKP cells.

This study investigated the effect of chronic hypertonicity on the OKP cell Na/H antiporter, encoded by Na/H exchanger 3 (NHE3). Chronic (48 h) increases in extracellular glucose, mannitol, or raffinose concentration caused a significant increase in Na/H antiporter activity, while increases in urea concentration were without effect. This effect was seen with changes in osmolality of only 20 mOsm/liter, a magnitude that is observed clinically in poorly controlled diabetes mellitus. Increases in mannitol concentration acutely inhibited and chronically stimulated Na/H antiporter activity. The increase in Na/H antiporter activity induced by hypertonic incubation was resistant to 10(-7) and 5 x 10(-6) M but inhibited by 10(-4) M ethylisopropyl amiloride, consistent with regulation of NHE3. In addition, hypertonicity increased total cellular and plasma membrane NHE3 protein abundance twofold, with only a small increase in NHE3 mRNA abundance. We conclude that chronic pathophysiologically relevant increases in tonicity lead to increases in NHE3 protein abundance and activity. This may be responsible for increased proximal tubule apical membrane Na/H antiporter activity in poorly controlled diabetes mellitus, which could then contribute to hypertension, glomerular hyperfiltration and diabetic nephropathy.

Amiloride↗
PubMed1998

Abnormal regulation of proximal tubule renin mRNA in the Dahl/Rapp salt-sensitive rat.

BACKGROUND: The precise pathogenesis of salt-sensitive hypertension in the Dahl rat is unknown. Abnormalities in renal hemodynamics and NaCl handling have been implicated, and may relate to changes in the activity of the intrarenal renin-angiotensin system. METHODS: Circulating, juxtaglomerular and intrarenal (glomerular and proximal tubular) renin were studied in Dahl/Rapp salt-sensitive and salt-resistant rats fed with a normal (0.5%) or high (4%) NaCl diet. Circulating and juxtaglomerular renin were assessed by measurement of plasma renin activity and renin secretory rates. Glomerular and proximal tubular renin mRNA were assessed by microdissection and quantitative competitive RT-PCR. RESULTS: Circulating and juxtaglomerular renin were suppressed by high dietary NaCl in salt-sensitive rats (plasma renin activity, 0.5%, 10.9 +/- 0.7 vs. 4%, 7.9 +/- 0.3 ng/ml/hr, P < 0.05; renin secretory rate, 0.5% 220 +/- 32 vs. 4%, 58 +/- 5 ng/mg/hr, P < 0.05). Glomerular renin mRNA was also suppressed by the higher salt diet in salt-sensitive animals (0.5%, 411 +/- 84 vs. 4%, 67 +/- 22 x 103 copies/glomerulus, P < 0.05). In contrast, proximal tubular renin was not suppressed by a high NaCl diet in salt-sensitive animals (0.5%, 13.9 +/- 2.7 vs. 4%, 12.1 +/- 3.6 x 103 copies/mm tubule, P = NS), but was suppressed in salt-resistant rats (0.5%, 9.5 +/- 2.8 vs. 4%, 3.2 +/- 1.2 x 103 copies/mm, P < 0. 05). CONCLUSIONS: Failure to suppress proximal tubular renin in response to high dietary NaCl may result in increased local generation of angiotensin II and enhanced proximal tubular NaCl absorption, and thereby contribute to the generation of salt sensitive hypertension.

Animals↗
PubMed1998

Effects of thyroid hormone on the neonatal renal cortical Na+/H+ antiporter.

The neonatal proximal tubule has a lower rate of bicarbonate absorption than that of adults. This is due, in part, to a lower rate of apical membrane Na+/H+ antiporter activity. The purpose of these studies was to examine if thyroid hormone could be a factor in the maturational increase in Na+/H+ antiporter activity. Hypothyroid (0.01% propylthiouracil in drinking water starting at day 14 gestation and throughout the postnatal period), euthyroid, and hyperthyroid (intraperitoneal triiodothyronine, 10 micrograms/100 g body wt, once daily on days 17 to 20 of postnatal life) rats were all studied at 21 days of life. Renal cortical brush border Na+/H+ antiporter activity was 453 +/- 24, 527 +/- 30 and 608 +/- 25 pmol/mg protein in the hypothyroid, euthyroid and hyperthyroid groups, respectively (P < 0.001). Hyperthyroid neonates had approximately twofold greater renal cortical NHE-3 mRNA abundance than euthyroid and hypothyroid neonates (P < 0.05). Brush border membrane NHE-3 protein abundance in hypothyroid and hyperthyroid neonates was one-third and twofold that of euthyroid 21-day-old rats, respectively (P < 0.001). These data are consistent with a potential role of thyroid hormone in the postnatal increase in Na+/H+ antiporter activity.

Animals↗
PubMed1998

Renal cortical mitochondrial aconitase is regulated in hypo- and hypercitraturia.

BACKGROUND: Chronic metabolic acidosis and K+ deficiency increase, while alkali feeding decreases proximal tubule citrate absorption and metabolism. The present studies examined the regulation of mitochondrial aconitase (m-aconitase), the first step in mitochondrial citrate metabolism, in these conditions. METHODS: Rats were fed appropriate diets, and m-aconitase activity and protein abundance measured. RESULTS: In chronic metabolic acidosis and chronic K+ deficiency, renal cortical m-aconitase activity was increased 17% and 43%, respectively. This was associated with respective 90% and 221% increases in renal cortical m-aconitase protein abundance. With chronic alkali feeding, there was a 12% decrease in renal cortical m-aconitase activity, associated with a 35% decrease in m-aconitase protein abundance. Hepatic m-aconitase activity was not regulated in a similar manner. There was no regulation of citrate synthase, the enzyme responsible for mitochondrial citrate synthesis. CONCLUSIONS: These studies demonstrate tissue specific chronic regulation of renal cortical m-aconitase activity and protein abundance, which likely contributes to the hypocitraturia and hypercitraturia seen in these conditions. As m-aconitase is the only step in citrate transport and metabolism found to be regulated in alkali feeding, its regulation likely plays a significant role in mediating the hypercitraturia seen in this condition.

Acidosis↗
PubMed1998

Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states.

Administration of pharmacologic doses of glucocorticoid in vivo increases renal proximal tubule apical membrane Na/H exchange and decreases Na/PO4 cotransport activity (1). Current data suggest that the NHE-3 and NaPi-2 proteins mediate significant fractions of proximal tubule apical membrane Na/H exchange and Na/PO4 cotransport, respectively. This study examines whether glucocorticoid excess or deficiency affects NHE-3 and NaPi-2 protein abundance and the intrarenal distribution of these transporters. Protein abundance of NHE-3 and NaPi-2 in control rats was compared to rats rendered glucocorticoid-deficient by bilateral adrenalectomy, and to rats receiving pharmacologic doses of dexamethasone using immunoblots and immunohistochemistry. Adrenalectomy had modest effects on NHE-3 protein abundance, but dexamethasone administration to either adrenalectomized or sham-operated rats significantly increased NHE-3 protein abundance in both the proximal tubule and thick ascending limb, but not the thin descending limb. Adrenalectomy increased NaPi-2 protein abundance in the proximal tubule, whereas dexamethasone administration dramatically suppressed NaPi-2 protein on the apical membrane in both adrenalectomized and sham-operated animals. No significant reciprocal increase in subapical NaPi-2 staining was seen in the dexamethasone-treated rats. The present study shows that glucocorticoids regulate proximal tubule apical membrane Na/H exchange and NaPi cotransport by changes in protein abundance of NHE-3 and NaPi-2, respectively.

Adaptor Protein Complex 3↗
PubMed1997

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.

Acidosis↗
PubMed1997

Sodium-hydrogen exchange in renal epithelia: mechanisms of acute regulation.

Five isoforms of sodium-hydrogen exchangers are present in the kidney. Specific isoforms serve specialized functions and are strategically located in polarized distributions in different nephron segments. This review updates understanding of the renal distribution and highlights some of the recent advances in understanding the mechanisms of acute regulation of sodium-hydrogen exchangers in the kidney.

Animals↗
PubMed1997

Intracellular acidosis activates c-Src.

The purpose of the present studies was to determine whether acidosis activates protein tyrosine kinase pathways. Incubation of MCT cells, a renal proximal tubule cell line, in acid media caused increased phosphotyrosine content of 60- to 70- and 120-kDa cytosolic proteins. Media acidification induced a twofold increase in c-Src activity that occurred within 30 s. Significant activation occurred with media pH changes as small as 0.07 pH unit accompanied by cell acidification of 0.06 pH unit. Sodium propionate addition, NH4Cl prepulse, and nigericin addition, maneuvers that decrease intracellular pH in the absence of changes in extracellular pH, activated c-Src. Significant activation by sodium propionate was seen with cell pH changes as small as 0.07 pH unit. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prevented c-Src activation by media acidification but did not prevent protein tyrosine phosphorylation. In summary, decreased intracellular pH activates c-Src. Acid activation of c-Src represents a novel mechanism of c-Src activation that may be relevant to many cellular responses to acidosis.

Acidosis↗
PubMed1997

Regulation of glomerular and proximal tubule renin mRNA by chronic changes in dietary NaCl.

Renal adaptations to chronic changes in dietary NaCl and extracellular fluid volume involve both glomerular and tubular mechanisms that result in preservation of glomerular filtration rate and modifications of renal tubular transport to secure external NaCl balance. Although the systemic renin-angiotensin system (RAS) mediates some of these responses, the possible contributions of local glomerular and proximal tubule RASs in these adaptations have not been examined. Thus, in this study, glomeruli and proximal tubules were microdissected from rats adapted to high (4.0%), normal (0.5%), or low (0.01%)-NaCl diets, and renin mRNA was measured using quantitative competitive reverse transcription-polymerase chain reaction. After 4 days of the diets, glomerular renin mRNA abundance was increased 100% by the low-NaCl diet (P < 0.05) and suppressed 50% (P < 0.01) by the high NaCl diet compared with controls. Renin mRNA in proximal tubules was stimulated 230% (P < 0.05) by the low-NaCl diet and tended to be suppressed (68% decrease, not significant) by the high-NaCl diet. When the high-NaCl diet was continued for 2 wk, proximal tubule renin mRNA was suppressed by 89% (P < 0.05). This study provides evidence that glomerular and proximal tubule renin transcript levels are regulated by chronic changes in dietary NaCl, suggesting that local RASs contribute to the renal adaptations in response to chronic alterations in NaCl.

Animals↗
PubMed1996

Adenosine triphosphate citrate lyase mediates hypocitraturia in rats.

Chronic metabolic acidosis increases proximal tubular citrate uptake and metabolism. The present study addressed the effect of chronic metabolic acidosis on a cytosolic enzyme of citrate metabolism, ATP citrate lyase. Chronic metabolic acidosis caused hypocitraturia in rats and increased renal cortical ATP citrate lyase activity by 67% after 7 d. Renal cortical ATP citrate lyase protein abundance increased by 29% after 3 d and by 141% after 7 d of acid diet. No significant change in mRNA abundance could be detected. Hypokalemia, which causes only intracellular acidosis, caused hypocitraturia and increased renal cortical ATP citrate lyase activity by 28%. Conversely, the hypercitraturia of chronic alkali feeding was associated with no change in ATP citrate lyase activity. Inhibition of ATP citrate lyase with the competitive inhibitor, 4S-hydroxycitrate, significantly abated hypocitraturia and increased urinary citrate excretion fourfold in chronic metabolic acidosis and threefold in K+-depletion. In summary, the hypocitraturia of chronic metabolic acidosis is associated with an increase in ATP citrate lyase activity and protein abundance, and is partly reversed by inhibition of this enzyme. These results suggest an important role for ATP citrate lyase in proximal tubular citrate metabolism.

ATP Citrate (pro-S)-Lyase↗
PubMed1996

Glucocorticoids regulate NHE-3 transcription in OKP cells.

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.

Animals↗