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.
It has been shown that short-term (hours) treatment with β-adrenergic agonists can stimulate lung liquid clearance via augmented Na+ transport across alveolar epithelial cells. This increase in Na+ transport with short-term β-agonist treatment has been explained by activation of the Na+ channel or Na+-K+-ATPase by cAMP. However, because the effect of sustained stimulation (days) with β-adrenergic agonists on the Na+ transport mechanism is unknown, we examined this question in cultured rat alveolar type II cells. Na+-K+-ATPase activity was increased in these cells by 10−4 M terbutaline in an exposure time-dependent manner over 7 days in culture. This increased activity was also associated with an elevation in transepithelial current that was inhibited by amiloride. The enzyme’s activity was also augmented by continuous treatment with dibutyryl-cAMP (DBcAMP) for 5 days. This increase in Na+-K+-ATPase activity by 10−4 M terbutaline was associated with an increased expression of α1-Na+-K+-ATPase mRNA and protein. β-Adrenergic agonist treatment also enhanced the expression of the α-subunit of the epithelial Na+ channel (ENaC). These increases in gene expression were inhibited by propranolol. Amiloride also suppressed this long-term effect of terbutaline and DBcAMP on Na+-K+-ATPase activity. In conclusion, β-adrenergic agonists enhance the gene expression of Na+-K+-ATPase, which results in an increased quantity and activity of the enzyme. This heightened expression is also associated with augmented ENaC expression. Although the cAMP system is involved, the inhibition of enhanced enzyme activity with amiloride suggests that increased Na+ entry at the apical surface plays a role in this process.
Chloride (Cl) channels have been proposed to play roles in lymphocyte functions including volume regulation and cellular cytotoxicity; however, direct studies of such channels in normal human lymphocytes are lacking. In the present study we describe a large conductance Cl channel observed in about 50% of excised, inside-out patches from normal human peripheral T lymphocytes. The channel has multiple conductance states with linear single-channel current-versus-voltage relationships in symmetrical Cl solutions. The most prevalent state is the largest, which has a conductance of about 365 pS. The channel closes in a voltage-dependent manner at both negative and positive potentials, but does not show voltage-dependent inactivation. The probability of opening is maximal between -15 mV and +15 mV and the voltage dependence is well described by two Boltzmann equations with half-maximal probabilities at -22.8 mV and +18.0 mV. The slopes of the voltage dependence suggest two gates in series with 5.7 and 9.6 equivalent charges. The channel was about 30 times more selective for Cl- than for Na+ or K+ under balanced osmolarity but less selective (approx. 11:1) under a large osmotic gradient. The single-channel conductance increased with Cl concentration with an apparent saturation at about 581 pS and a Michaelis-Menten constant of about 120 mM. The selectivity sequence among anions, determined from changes in reversal potential was: I- greater than NO3- greater than Br-, Cl- greater than F-, isethionate, HCO3- greater than SO4(2-) greater than gluconate, propionate greater than aspartate much greater than Na+, K+ and was apparently the same for subconductance states.(ABSTRACT TRUNCATED AT 250 WORDS)
The patch-clamp technique was applied to study ion conductances in various configurations of the nuclear envelope of non-enzyme-treated red beet (Beta vulgaris L.) nuclei. With excised patches a non-selective cation channel was observed, that was activated by micromolar concentrations of Ca2+ on the nucleoplasmic side of the envelope. The channel activity was also voltage-dependent and the voltage threshold of channel activation changed with the nucleoplasmic Ca2+ concentration. The most prominent conductance level was 110+/-22 pS with 150 mM KCl in the bath and pipette. The channel was permeable to small cations: permeabilities relative to K+ were PK congruent with PNa=1, PCs=0.3, but PCl=0.09. Calcium ions also permeated the channel with PCa=0.43, estimated from reversal potential, or 0.14, estimated from conductance ratio. Zn2+ (1 mM) when applied to the cytoplasmic side of the envelope blocked the channel activity completely, while amiloride (2 mM) reduced the channel current by 86% from the nucleoplasmic side. The properties of the whole-nucleus current (voltage-, time- and Ca2+-dependence) paralleled those observed with excised patches. The channel may provide a Ca2+-regulated pathway for passive diffusion of cations across the nuclear envelope and thus may play an important role in Ca2+-dependent nuclear processes ranging from gene transcription to apoptosis.
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