The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells

Analysis of membrane currents recorded from hormone-deprived H441 cells showed that the membrane potential (V(m)) in single cells (approximately -80 mV) was unaffected by lowering [Na+]o or [Cl(-)]o, indicating that cellular Na+ and Cl(-) conductances (GNa and GCl, respectively) are negligible. Although insulin (20 nM, approximately 24 h) and dexamethasone (0.2 microM, approximately 24 h) both depolarized Vm by approximately 20 mV, the response to insulin reflected a rise in GCl mediated via phosphatidylinositol 3-kinase (PI3K) whereas dexamethasone acted by inducing a serum- and glucocorticoid-regulated kinase 1 (SGK1)-dependent rise in GNa. Although insulin stimulation/PI3K-P110 alpha expression did not directly increase GNa, these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGK1-induced GNa in single cells discriminated poorly between Na+ and K+ (PNa/PK approximately 0.6), was insensitive to amiloride (1 mM), but was partially blocked by LaCl3 (La3+; 1 mM, approximately 80%), pimozide (0.1 mM, approximately 40%), and dichlorobenzamil (15 microM, approximately 15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10 microM), selective GNa that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective GNa (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGK1 and subject to PI3K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of Na+ selectivity and amiloride sensitivity.     

Dexamethasone blocks adaptive increase of Na+-Pi cotransport in renal brush border membrane elicited by thyroid hormone

Dexamethasone administered to rats blocks and/or reverses adaptive increases in the rate of Na+-Pi cotransport, and also in the Na+-dependent binding of [14C]-phosphonoformic acid (PFA) by renal brush border membrane (BBM) vesicles elicited by thyroid hormone (T3). In contrast, dexamethasone had no effect on Na+-independent binding of [14C]-phosphonoformic acid, on Na+-dependent transport of D-glucose or on Na+-dependent binding of phlorizin by BBMV which indicates that its inhibitory effect is specific for Na+-Pi cotransport system of BBM. These findings suggest that glucocorticoids antagonize T3-elicited adaptive enhancement of Na+-Pi cotransport in renal proximal tubules by blocking the T3-stimulated de novo synthesis of Na+-Pi symporters and/or their insertion into BBM.     

Maintenance of brain thyroid hormone level during peripheral hypothyroid condition in adult rat

Thyroid hormones are essential for normal functioning of adult mammalian brain. The present investigation deals with the understanding of the time course of thyroid hormone homeostasis in adult rat brain. Animals were rendered hypothyroid by PTU injections (2 mg/100 g bw) for 30 consecutive days. Serum and synaptosomal T3/T4 content, synaptosomal AChE and Na+-K+-ATPase activities were determined on alternate days. While serum T4 level initially increased on the second day compared to control, serum T3 declined in a triphasic pattern; the first phase lasting from the second day to the 6th day, the second phase ended on the 14th day and last phase continued till the 30th day. Cerebro-cortical synaptosomal T3 level increased on the 2nd day from the control, attained a peak on the 4th day, remained stable until the 18th day, and abruptly declined on the 20th day. Synaptosomal T4 content remained negligible or undetected throughout. Synaptosomal membrane Na+-K+-ATPase and AChE activity exhibited an inverse relationship during the experimental regime, being much more prominent on the 2nd, 18th and 20th day coinciding with the variations in brain T3 level. Thus, the study identifies the onset of central homeostasis between the first and second day, its continuation for about 16-18 days and its termination between the 18th and 20th day.     

Quercetin and NPPB-induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl- secretion over 30-fold due to activation of the Na+/K+/2Cl- cotransporter and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC), and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity, and alpha-ENaC mRNA expression was diminished by 24h-pretreatment with quercetin (an activator of Na+/K+/2Cl- cotransporter participating in Cl- entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl- channel participating in Cl- release from the cytosolic space), while 24h-pretreatment with bumetanide (a blocker of Na+/K+/2Cl- cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of the Na+/K+/2Cl- cotransporter, and that modification of activity of Cl- transporter/channel participating in the transepithelial Cl- secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport).     

Nuclear activation by thyroid hormone in liver of Singi fish: changes in different ion-specific adenosine triphosphatases activities

Various ion-dependent (Na+K+, Ca++ and Mg++) ATPases activities in liver cell nuclear membrane have been determined after a single injection of different doses (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 and 4 micrograms/g) of L-triiodothyronine (T3) in Singi fish, Heteropneustes fossilis Bloch. Administration of T3 at a minimum effective dose of 0.05 micrograms upto 4 micrograms/g induced a rise (14 to 43% over control value) in the Na+K+-ATPase activity in a dose-dependent fashion maximum upto 1 microgram/g dose, whereas Ca++-ATPase showed a dose-dependent increase (20 to 43% over control) with 0.25-1 microgram/g of T3, although the increase in the respective enzyme activity was maintained upto 4 micrograms/g of T3 dose. Mg++-ATPase activity in liver cell nuclear membrane was found to be increased at 1 microgram-4 micrograms/g of T3 dose, showing a similar magnitude of increase (7% over the control value) with these doses of T3. Other doses of T3 (0.01 and 0.025 micrograms/g) were ineffective in altering the different ion-specific ATPase activity. Treatment of Singi fish with thiourea (1 mg/ml) for 30 days caused a significant fall in Na+K+, Ca++ and Mg++-ATPase activities upto 21%, 17% and 5%, respectively, below the euthyroid control level. A single injection of T3 at the dose of 1 microgram/g in the hypothyroid fish raised the Na+K+ and Ca++-ATPase activities to about 36% over the control value, and the Mg++-ATPase activity was restored to only the control level. Thus a dose-dependent nuclear effect of T3 is evident from the present investigation.     

Modulation of rat distal colonic brush-border membrane Na+-H+ exchange by dexamethasone: role of lipid fluidity

Earlier studies by our laboratory have suggested a relationship between an amiloride-sensitive Na+-H+ exchange process and the physical state of the lipids of rat colonic brush-border membrane vesicles. To further assess this possible relationship, a series of experiments were performed to examine the effect of dexamethasone administration (100 micrograms/100 g body wt. per day) subcutaneously for 4 days on Na+-H+ exchange, lipid composition and lipid fluidity of rat distal colonic brush-border membrane vesicles. The results of these studies demonstrate that dexamethasone treatment significantly: (1) increased the Vmax of the Na+-H+ exchange without altering the Km for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange. 22Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across dexamethasone-treated brush-border membrane vesicles; (2) increased the lipid fluidity of treated-membrane vesicles compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using three different lipid-soluble fluorophores; and (3) increased the phospholipid content of treated-membrane vesicles thereby, decreasing the cholesterol/phospholipid molar ratio of treated compared to control preparations. This data, therefore, demonstrates that dexamethasone administration can modulate amiloride-sensitive Na+-H+ exchange in rat colonic distal brush-border membrane vesicles. Moreover, it adds support to the contention that a direct relationship exists between Na+-H+ exchange activity and the physical state of the lipids of rat colonic apical plasma membranes.     

Regulation of epithelial Na+ channels by adrenal steroids: mineralocorticoid and glucocorticoid effects

Epithelial Na+ channels (ENaC) can be regulated by both mineralocorticoid and glucocorticoid hormones. In the mammalian kidney, effects of mineralocorticoids have been extensively studied, but those of glucocorticoids are complicated by metabolism of the hormones and cross-occupancy of mineralocorticoid receptors. Here, we report effects of dexamethasone, a synthetic glucocorticoid, on ENaC in the rat kidney. Infusion of dexamethasone (24 μg/day) for 1 wk increased the abundance of αENaC 2.26 ± 0.04-fold. This was not accompanied by an induction of Na+ currents (I(Na)) measured in isolated split-open collecting ducts. In addition, hormone treatment did not increase the abundance of the cleaved forms of either αENaC or γENaC or the expression of βENaC or γENaC protein at the cell surface. The absence of hypokalemia also indicated the lack of ENaC activation in vivo. Dexamethasone increased the abundance of the Na+ transporters Na+/H+ exchanger 3 (NHE3; 1.36 ± 0.07-fold), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2; 1.49 ± 0.07-fold), and Na-Cl cotransporter (NCC; 1.72 ± 0.08-fold). Surface expression of NHE3 and NCC also increased with dexamethasone treatment. To examine whether glucocorticoids could either augment or inhibit the effects of mineralocorticoids, we infused dexamethasone (60 μg/day) together with aldosterone (12 μg/day). Dexamethasone further increased the abundance of αENaC in the presence of aldosterone, suggesting independent effects of the two hormones on this subunit. However, I(Na) was similar in animals treated with dexamethasone+aldosterone and with aldosterone alone. We conclude that dexamethasone can occupy glucocorticoid receptors in cortical collecting duct and induce the synthesis of αENaC. However, this induction is not sufficient to produce an increase in functional Na+ channels in the apical membrane, implying that the abundance of αENaC is not rate limiting for channel formation in the kidney.     

Modulation of alpha-ENaC and alpha1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells

cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.     

Evidence for an intracellular site of action in the heart for two hydrophobic cardiac steroids

Although cardiac steroids (CS) have long been used to treat cardiac insufficiency, the mechanism(s) of action of these agents remain open to question. While many results indicate that inhibition of Na+,K+-ATPase underlies both the therapeutic and toxic actions of CS, other studies suggest that actions on the SR membrane system may be important. We used two experimental approaches and measurements of left ventricular diastolic pressure (LVDP) in isolated guinea pig hearts to test whether CS had an intracellular site of action. In the first approach, we compared the inotropic effects of a hydrophilic CS, ouabain, and a hydrophobic CS, digitoxin, after the activity of the Na+ pump was reduced by perfusing hearts with solutions maintained at 5 degrees C. Under these conditions, exposure of hearts to 1 microM ouabain for 60 min did not increase LVDP above control levels. In contrast, an equi-effective concentration of digitoxin (0.3 microM) increased LVDP by 40 +/- 8.5% (p < 0.01) over pre-drug control levels. In the second experimental approach, we compared the inotropic effects of ouabain and digitoxin in the presence of rapid-cooling contractures (RCC), which result in the release of SR Ca2+. Hearts were perfused with Tyrode solution or Tyrode solution containing either digitoxin (0.3 microM) or ouabain (1 microM) for 180 sec, rapidly cooled and the RCC responses were analyzed. Compared to RCC elicited in Tyrode solution alone, or in Tyrode solution containing ouabain, RCC in the presence of digitoxin reached peak amplitudes more rapidly, but elicited reduced peak amplitude values. Based on these findings, we suggest that: 1) the ability of the hydrophobic CS, digitoxin, but not the hydrophilic CS, ouabain, to produce a positive inotropic effect at 5 degrees C, when the activity of the Na+ pump is markedly reduced, is consistent with a mechanism other than Na+ pump inhibition and involves an intracellular location; and 2) the diminished RCC observed in the presence of the hydrophobic CS, digitoxin, indicate that this alternative mechanism may involve effects on the SR Ca2+ release channel.     

Glucocorticoids stimulate the activity of large-conductance Ca2+ -activated K+ channels in pituitary GH3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca2+ -activated K+ current (IK(Ca)) with an EC50 value of 21 +/- 5 microM. Dexamethasone-induced increase in IK(Ca) density was suppressed by paxilline (1 microM), yet not by glibenclamide (10 microM), pandinotoxin-Kalpha (1 microM) or mifepristone (10 microM). Paxilline is a blocker of large-conductance Ca2+ -activated K+ (BKCa) channels, while glibenclamide and pandinotoxin-Kalpha are blockers of ATP-sensitive and A-type K+ channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 microM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BKCa -channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca2+ -sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 microM) also increased BKCa -channel activity. Dexamethasone-mediated stimulation of IK(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Dexamethasone and thyroid hormone pretreatment upregulate alveolar epithelial fluid clearance in adult rats.

The in vivo effect of 48-h glucocorticoid and thyroid hormone 3,3', 5-triiodine-L-thyronine (T(3)) pretreatment on alveolar epithelial fluid transport was studied in adult rats. An isosmolar 5% albumin solution was instilled, and alveolar fluid clearance was studied for 1 h. Compared with controls, dexamethasone pretreatment increased alveolar fluid clearance by 80%. T(3) pretreatment stimulated alveolar fluid clearance by 65%, and dexamethasone and T(3) had additive effects (132%). Propranolol did not inhibit alveolar fluid clearance in either group, indicating that stimulation was not secondary to endogenous beta-adrenergic stimulation. With the use of bromodeoxyuridine in vivo labeling, there was no evidence of cell proliferation. Alveolar fluid clearance was partially inhibited by amiloride in all groups. Fractional amiloride inhibition was greater in dexamethasone- and dexamethasone-plus-T(3)-pretreated rats than in control animals, but less in T(3)-pretreated rats. In summary, pretreatment with dexamethasone, T(3), or both in combination upregulate in vivo alveolar fluid clearance similarly to short-term beta-adrenergic stimulation. The effects are mediated partly by increased amiloride-sensitive Na(+) transport, because the stimulated alveolar fluid clearance was more amiloride sensitive than in control rats. These observations may have clinical relevance because glucocorticoid therapy is commonly used with acute lung injury.     

Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase

Low density lipoproteins (LDL) inhibit the Na+/H+ antiport and thereby sensitize platelet towards agonist. However, mechanisms underlying the suppressing effect of LDL on Na+/H+ exchange are unclear. We here show that the lowering of intracellular pH and the suppression of the sodium propionate-induced Na+/H+ exchange in the presence of LDL are abolished by SKF86002, a selective inhibitor of p38MAP kinase (p38MAPK). The inhibitory effect of LDL on Na+/H+ exchange was mimicked by H2O2, which directly activates p38MAPK. Exposure of platelets to LDL or H2O2 led to phosphorylation of p38MAPK, its upstream regulator MAP kinase kinase 3/6 (MKK 3/6), and its downstream target heat shock protein 27 (HSP27), and this effect was abrogated in SKF86002-pretreated platelets. In addition, both LDL and H2O2 produced the SKF86002-sensitive phosphorylation of an oligopeptide encompassing p38MAPK phosphorylation sequence derived from NHE-1, a major Na+/H+ exchanger in platelets. We further show that the sensitizing effects of LDL on the thrombin-induced platelet activation, as reflected by aggregation and granule secretion, are abolished in cells pretreated with SKF86002. We conclude that activation of p38MAPK is required for the inhibitory effect of LDL on Na+/H+ antiport and thereby for LDL-dependent sensitization in human platelets.     

Thyroid hormone-induced changes in different ion-specific adenosine triphosphatase activities in liver of Singi fish Heteropneustes fossilis (Bloch)

A single injection of different doses of T3 (0.5, 5, 20, and 50 micrograms/g) to Singi fish caused an increase in Na+K+-ATPase activity in crude liver homogenate in a dose-dependent non-linear fashion on the 3rd d. Ca++- and Mg++-ATPase activity increased only with 20 and 50 micrograms/g of T3. Lowering the dose of T3 to 0.1 microgram and 0.25 microgram/g in a single injection had not effect on these enzyme activities. TETRAC (1, 2, and 4 micrograms/g) and TRIAC (2 and 4 micrograms/g) in a single injection enhanced the activities of Na+K+-ATPase, but Ca++- and Mg++-ATPase activities remained unchanged on the 3rd d. Immersion of Singi fish in thiourea-containing medium (1 mg/ml) for 30 d caused reduction in Na+K+-ATPase activity, but Ca++- and Mg++-ATPase activity remained unaltered. The reduced level of Na+K+-ATPase activity in the thiourea-treated hypothyroid fish was recovered and even brought above the control level by a single injection of T3 at the dose of 0.5 microgram/g. Differential sensitivity of various ion-specific ATPases to T3 in liver of Singi fish is thus documented.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.