Epithelial Na(+) channels (ENaC) are located in alveolar cells and are important in β(2)-adrenergic receptor-mediated lung fluid clearance through the removal of Na(+) from the alveolar airspace. Previous work has demonstrated that genetic variation of the alpha subunit of ENaC at amino acid 663 is important in channel function: cells with the genotype resulting in alanine at amino acid 663 (A663) demonstrate attenuated function when compared to genotypes with at least one allele encoding threonine (T663, AT/TT). We sought to determine the influence of genetic variation at position 663 of ENaC on exhaled Na(+) in healthy humans. Exhaled Na(+) was measured in 18 AA and 13 AT/TT subjects (age=27±8 years vs. 30±10 years; ht.=174±12 cm vs. 171±10 cm; wt.=68±12 kg vs. 73±14 kg; BMI=22±3 kg/m(2) vs. 25±4 kg/m(2), mean±SD, for AA and AT/TT, respectively). Measurements were made at baseline and at 30, 60 and 90 min following the administration of a nebulized β(2)-agonist (albuterol sulfate, 2.5 mg diluted in 3 ml normal saline). The AA group had a higher baseline level of exhaled Na(+) and a greater response to β(2)-agonist stimulation (baseline=3.1±1.8 mmol/l vs. 2.3±1.5 mmol/l; 30 min-post=2.1±0.7 mmol/l vs. 2.2±0.8 mmol/l; 60 min-post=2.0±0.5 mmol/l vs. 2.3±1.0 mmol/l; 90 min-post=1.8±0.8 mmol/l vs. 2.6±1.5 mmol/l, mean±SD, for AA and AT/TT, respectively, p
The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI).
We examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.
Although thrombin is best known for its role in blood coagulation, it has been reported to change the activity of ion motive ATPases in some tissues. In the present study, experiments were conducted to determine the influence of thrombin on active sodium-potassium transport in porcine lenses.
1. The concentrations of sodium, potassium and chloride in frog and bovine lenses showed a normal intracellular ion distribution with the sum of the internal cations approximately equal to the external sum. In the cephalopod lens, however, the sum inside was much lower than that outside.2. The membrane potentials of frog, Sepiola and bovine lenses were -63, -63 and -23 mV respectively. A comparison of the electrical data with the Nernst potentials predicted from ion concentration data indicated that sodium and chloride ions as well as potassium contributed to the membrane potential in frog and bovine. In contrast, the membrane and Nernst potentials for potassium were equal in Sepiola.3. Substituting potassium for sodium in the external medium depolarized lens potentials in all three species. Estimates of the relative permeabilities of sodium, potassium and chloride were obtained by fitting the Goldman-Hodgkin-Katz equation to the potential data.4. The potassium permeability was determined directly by (42)K efflux measurements and values of 2.99, 9.83 and 3.13 (x (-8) m sec(-1)) were obtained for frog, Sepiola and bovine lenses respectively.5. The effect of raising external potassium on the efflux rate constant was determined and there was reasonable agreement between experiment and theory (Kimizuka-Koketsu) in frog and bovine lenses, but the Sepiola data indicated that the potassium permeability decreased by a factor of 2.6 when the external potassium was raised from 10 to 120 mM-K+.6. The measured specific conductances, obtained using two internal micro-electrodes, were 7.7, 15.9 and 9.9 (Sm(-2)) for frog, cephalopod and bovine lenses respectively. These data compare with computed values (Kimizuka-Koketsu theory) of 7.5, 14.1 and 17.2 (Sm(-2)).7. The effect of increasing external potassium on the conductance was also tested and there was good agreement between experiment and theory (assuming constant permeabilities) only in the amphibian lens. However, when the cephalopod data were corrected assuming a 2.6-fold decrease in P(K) for a twelvefold increase in potassium, then there was excellent agreement between experiment and theory.8. The bovine measured conductances were much lower than the theoretical values throughout the range of external potassium concentrations and several explanations were proposed to account for the discrepancies.