M. J. Stutts

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell.

Chloride secretory response of cystic fibrosis human airway epithelia. Preservation of calcium but not protein kinase C- and A-dependent mechanisms.

Because the defect in Cl- secretion exhibited by cystic fibrosis (CF) epithelia reflects regulatory rather than conductive abnormalities of an apical membrane Cl- channel, we investigated the role of different regulatory pathways in the activation of Cl- secretion in freshly excised normal and CF nasal epithelia mounted in Ussing chambers. A beta agonist (isoproterenol [ISO]), a Ca2+ ionophore (A23187), and a phorbol ester (PMA) were all effective Cl- secretagogues in normal human nasal epithelia. Agonist addition studies indicated that ISO and PMA but not A23187 may share a common regulatory pathway. In contrast, only A23187 induced Cl- secretion in CF epithelia. Bradykinin raised cytosolic Ca2+ and induced Cl- secretion in both normal and CF tissues, indicating that receptor gated Ca2+ dependent Cl- secretory mechanisms were preserved in CF. The defective Cl- secretory response in CF epithelia to ISO and PMA did not reflect abnormalities in cAMP-dependent (A) and phospholipid Ca2+-dependent (C) kinase activities. We conclude that (a) a Ca2+-sensitive mechanism for regulating Cl- secretion is maintained in CF airway epithelia, and (b) a regulatory pathway shared by two distinct protein kinases is defective in CF, indicating that the CF genetic lesion is not tightly coupled to a single (e.g., cAMP dependent) regulatory mechanism.

Abnormal apical cell membrane in cystic fibrosis respiratory epithelium. An in vitro electrophysiologic analysis.

The transepithelial chloride permeability of airway and sweat ductal epithelium has been reported to be decreased in patients with cystic fibrosis (CF). In the present study, we investigated whether the airway epithelial defect was in the cell path by characterizing the relative ion permeabilities of the apical membrane of respiratory epithelial cells from CF and normal subjects. Membrane electric potential difference (PD) and the responses to luminal Cl- replacement, isoproterenol, and amiloride were measured with intracellular microelectrodes. The PD across the apical barrier was smaller for CF (-11 mV) than normal (-29 mV) epithelia whereas the PD across the basolateral barrier was similar, (-26 and -34 mV respectively). In contrast to normal nasal epithelium, the apical membrane in CF epithelia was not Cl- permselective and was not responsive to isoproterenol. Amiloride, a selective Na+ channel blocker, induced a larger apical membrane hyperpolarization and a greater increase in transepithelial resistance in CF epithelia. Both reduced apical cell membrane Cl- conductance and increased Na+ conductance appear to contribute to the abnormal function of respiratory epithelia of CF patients.

Oxygen consumption and ouabain binding sites in cystic fibrosis nasal epithelium

ABSTRACT. Ion transport by the epithelium lining the airways of patients with cystic fibrosis (CF) is characterized by a raised transepithelial PD and an increased amiloride sensitivity (1). These properties could arise from normal sodium transport across an epithelium with decreased cell chloride permeability and limited chloride secretion. Alternatively, a higher than normal rate of sodium absorption could contribute to these abnormalities. We investigated the latter possibility by measuring oxygen consumption and specific ouabain binding of CF and atopic polyp epithelia and normal turbinate epithelium. Tissue from CF patients consumed oxygen at a rate that was two to three times that of non-CF tissues and had 60% more ouabain binding sites than non-CF epithelium. These results are not consistent with an isolated defect in chloride permeability but support recent findings that the sodium conductance of the apical cell membrane and net sodium absorption by CF nasal epithelium are greater than those of non-CF nasal epithelium.

Effects of ammonium and nitrate salts on ion transport across the excised canine trachea

The effects of ammonium and sodium salts of nitrate, nitrite, and chloride on bioelectric properties and unidirectional Na and Cl fluxes across canine trachea were examined. Na salts of nitrate and nitrite (100 mmol/liter) had little effect. NH4NO3 or NH4Cl (100 mmol/liter) reduced short-circuit current (Isc), Na+ absorption, and Cl− secretion. Exposure of the luminal surface led to approximately 50% reductions in Isc and potential difference (PD). Submucosal exposure to either salt completely abolished Isc, PD, and ion transport. The effects of luminal or submucosal exposure were reversed by washing the tissue with Krebs bicarbonate Ringer solution. In Hepesbuffered solutions, submucosal exposure induced a poorly reversible loss of ion transport. Furthermore, 30 mmol/liter NH4 salt solutions buffered by Hepes and 100 mmol/liter NH4+ in bicarbonate Ringer were equipotent. The potency of luminal NH4+ solutions was unaffected by solution buffer. We propose that the NH3 in NH4+ solutions alkalinizes the epithelial cytoplasm. The extent of alkalinization may depend on the NH4+ permeability of the surface exposed to the salt. NH4+ entry across the luminal cell membrane could limit the alkalinization of cell contents by dissolved NH3.

Activation of chloride conductance induced by potassium in tracheal epithelium

The effects of partial replacement of bathing solution sodium by potassium on potential difference, conductance and ion transport of canine tracheal epithelium were studied in Ussing chambers. Whereas up to 100 mmol/l mucosal K+ had no effect, raised serosal [K+] induced a concentration dependent decrease in transepithelial potential difference and increase in conductance. When serosal K+ was 100 mmol/l, the potential difference fell 30 mV to 1.1±1.0 mV and conductance rose 4.5 mS/cm2 to 6.6 ±0.9 mS/cm2. Seventy-five percent of the K+ induced conductance required Cl− (120 mmol/l) in the luminal bathing solution. Unidirectional Cl− fluxes were increased by raised serosal K+ but14C-mannitol permeability was unchanged. The increased unidirectional Cl− flux induced by high K+ exposure was inhibited by luminal exposure to diphenylamine-2-carboxylic acid (DPC) or other chloride channel blockers, but was not inhibited by loop diuretics. These results suggest that in the presence of 100 mmol/l serosal K+ the transcellular chloride conductance of tracheal epithelium was increased. Increased chloride conductance of the apical cell membrane contributed to the raised transcellular permeability, but the route across the basolateral cell membrane was not identified.

Effects of formaldehyde on bronchial ion transport

The functions of the epithelium that lines mammalian airways are potential targets for the toxic effects of reactive chemicals such as formaldehyde. We examined the effects of formaldehyde on bioelectric properties and ion permeation of excised canine and human bronchial epithelium. Concentration-dependent reductions in short-circuit current were induced in both tissues. Sodium absorption and transcellular chloride fluxes across canine bronchial epithelium were inhibited by 65 and 35%, respectively, by 10(-3) M formaldehyde. The QO2 of isolated dog bronchial epithelial cells was reduced by 32% by 10(-3) M formaldehyde. These results are consistent with an action of formaldehyde on cellular ion permeability but effects secondary to metabolic inhibition cannot be dismissed.