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TGF-β1 is a major mediator of airway tissue remodelling during atopic asthma and affects tight junctions (TJs) of airway epithelia. However, its impact on TJs of ciliated epithelia is sparsely investigated. Herein we elaborated effects of TGF-β1 on TJs of primary human bronchial epithelial cells. We demonstrate that TGF-β1 activates TGF-β1 receptors TGFBR1 and TGFBR2 resulting in ALK5-mediated phosphorylation of SMAD2. We observed that TGFBR1 and -R2 localize specifically on motile cilia. TGF-β1 activated accumulation of phosphorylated SMAD2 (pSMAD2-C) at centrioles of motile cilia and at cell nuclei. This triggered an increase in paracellular permeability via cellular redistribution of claudin 3 (CLDN3) from TJs into cell nuclei followed by disruption of epithelial integrity and formation of epithelial lesions. Only ciliated cells express TGF-β1 receptors; however, nuclear accumulations of pSMAD2-C and CLDN3 redistribution were observed with similar time course in ciliated and non-ciliated cells. In summary, we demonstrate a role of motile cilia in TGF-β1 sensing and showed that TGF-β1 disturbs TJ permeability of conductive airway epithelia by redistributing CLDN3 from TJs into cell nuclei. We conclude that the observed effects contribute to loss of epithelial integrity during atopic asthma.

The lung is the interface between air and blood where the exchange of oxygen and carbon dioxide occurs. The surface liquid that is directly exposed to the gaseous compartment covers both conducting airways and respiratory zone and forms the air-liquid interface. The barrier that separates this lining fluid of the airways and alveoli from the extracellular compartment is the pulmonary epithelium. The volume of the lining fluid must be kept in a range that guarantees an appropriate gas exchange and other functions, such as mucociliary clearance. It is generally accepted that this is maintained by balancing resorptive and secretory fluid transport across the pulmonary epithelium. Whereas osmosis is considered as the exclusive principle of fluid transport in the airways, filtration may contribute to alveolar fluid accumulation under pathologic conditions. Aquaporins (AQP) facilitate water flux across cell membranes, and as such, they provide a transcellular route for water transport across epithelia. However, their contribution to near-isosmolar fluid conditions in the lung still remains elusive. Herein, we discuss the role of AQPs in the lung with regard to fluid homeostasis across the respiratory epithelium.

A commonly used technique to investigate strain-induced responses of adherent cells is culturing them on an elastic membrane and globally stretching the membrane. However, it is virtually impossible to acquire microscopic images immediately after the stretch with this method. Using a newly developed technique, we recorded the strain-induced increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in rat primary alveolar type II (ATII) cells at an acquisition rate of 30ms and without any temporal delay. We can show that the onset of the mechanically induced rise in [Ca(2+)](c) was very fast (<30 ms), and Ca(2+) entry was immediately abrogated when the stimulus was withdrawn. This points at a direct mechanical activation of an ion channel. RT-PCR revealed high expression of TRPV2 in ATII cells, and silencing TRPV2, as well as blocking TRPV channels with ruthenium red, significantly reduced the strain-induced Ca(2+) response. Moreover, the usually homogenous pattern of the strain-induced [Ca(2+)](c) increase was converted into a point-like response after both treatments. Also interfering with actin/myosin and integrin binding inhibited the strain-induced increase of [Ca(2)](c). We conclude that TRPV2 participates in strain-induced Ca(2+) entry in ATII cells and suggest a direct mechanical activation of the channel that depends on FAs and actin/myosin. Furthermore, our results underline the importance of cell strain systems that allow high temporal resolution.

A thin layer of airway surface liquid (ASL) lines the entire surface of the lung and is the first point of contact between the lung and the environment. Surfactants contained within this layer are secreted in the alveolar region and are required to maintain a low surface tension and to prevent alveolar collapse. Mucins are secreted into the ASL throughout the respiratory tract and serve to intercept inhaled pathogens, allergens and toxins. Their removal by mucociliary clearance (MCC) is facilitated by cilia beating and hydration of the ASL by active ion transport. Throughout the lung, secretion, ion transport and cilia beating are under purinergic control. Pulmonary epithelia release ATP into the ASL which acts in an autocrine fashion on P2Y(2) (ATP) receptors. The enzymatic network describes in Chap. 2 then mounts a secondary wave of signaling by surface conversion of ATP into adenosine (ADO), which induces A(2B) (ADO) receptor-mediated responses. This chapter offers a comprehensive description of MCC and the extensive ramifications of the purinergic signaling network on pulmonary surfaces.

Proximal tubular cells of the frog (Rana esculenta) kidney were fused within an isolated tubule portion to giant cells according to the polyethylene-glycol fusion method. Cell membrane potentials (Vm) were measured while cells were superfused with various experimental solutions. Rapid concentration step-changes of different ions allowed to calculate the respective transference numbers (tion). In some experiments the specific cell membrane resistances (Rm) were evaluated by measuring Vm induced by short current pulses injected into the cell with a second electrode. The experiments reveal: i) Fused cells of the proximal tubule exhibit a Vm of -49.5 +/- 1.6 mV (n = 65). ii) Addition of glucose to the perfusate yields a transient depolarization, consistent with a rheogenic Na/glucose cotransport system. iii) In absence of organic substrates the whole cell membrane conductance is made up of K+ and HCO3-. iv) There is a positive relationship between Vm and tK+ and a negative relationship between Vm and tHCO3-. v) HCO3--induced Vm changes are attenuated or abolished when Na+ is replaced with choline+, consistent with a rheogenic Na+/HCO3- cotransport system. vi) Replacement of Na+ by choline+ depolarizes Vm and increases Rm by about 50%; addition of 3 mmol/liter Ba2+ to the Na+-free perfusate increases Rm by about 58% compared to the initial control value. vii) There is no measurable cell membrane Cl- conductance. We conclude that fused cells of proximal tubule exert both luminal and peritubular membrane properties. In absence of organic substrates the cell membrane potential is determined by the HCO3- and K+ transport systems.

Experiments were performed in the isolated perfused kidney of K+ adapted Rana pipiens to investigate the relationship between luminal K+ conductance and H+ transport in cells of the diluting segment. Inhibition of luminal Na+/H+ exchange by amiloride or by omission of luminal Na+ blocked luminal K+ conductance. Acidification of the kidney perfusate by elevation of pCO2 also reduced luminal K+ conductance. This effect could be prevented by furosemide. Since the steepest transcellular Na+ potential difference, directed from the lumen into the cell, is found when luminal Na+/Cl-/K+ cotransport is inhibited by furosemide, we conclude that luminal Na+/H+ exchange is most efficient at these conditions and thus could attenuate intracellular acidification.

In the isolated-perfused frog (Rana pipiens) kidney the question of whether transepithelial transport of Ca2+ is a passive voltage driven process or involves active mechanisms was investigated. With conventional and ion-sensitive microelectrodes transepithelial electrical and electrochemical potential differences were measured. Luminal activities and transepithelial net fluxes of Ca2+ and Cl- were evaluated. Different transepithelial electrical voltages in a wide range (+20 to -4 mV) were generated by "chemical voltage clamping" and the dependence of Ca2+ net fluxes on these voltages investigated. The hormonal control of both Cl- and Ca2+ transport was studied by evaluating the effect of the cell-permeable cAMP analogue, db-cAMP and of the adenylate cyclase stimulator, forskolin. The experiments reveal that: (a) Ca2+ is reabsorbed along the diluting segment of frog kidney. (b) Ca2+ reabsorption is inhibited by furosemide because of the elimination of the transepithelial voltage. (c) There is a direct relationship between transepithelial voltage and Ca2+ reabsorption. (d) Neither Cl- nor Ca2+ reabsorption are affected by db-cAMP or forskolin. We conclude that Ca2+ reabsorption is passive, driven by the lumen-positive transepithelial voltage. It most likely occurs via the paracellular shunt pathway.

The mechanism of HCO3- transport was studied applying microelectrodes in "giant" cells fused from single epithelial cells of the diluting segment of frog kidney. A sudden increase of extracellular HCO3- concentration from 10 to 20 mmol/l at constant pH hyperpolarized the cell membrane potential of the fused cell. This cell-voltage response was totally abolished by 10(-3) mol/l SITS and significantly reduced by 10(-4) mol/l acetazolamide or by omission of Na+ from the extracellular perfusate. Removal of Na+ from the perfusate caused a transient depolarization. Reapplication of Na+ induced a transient hyperpolarization. 10(-3) mol/l SITS abolished the cell-voltage response to removal and reapplication of Na+. In the intact diluting segment of the isolated perfused frog kidney peritubular perfusion of 10(-4) mol/l acetazolamide reduced the limiting transepithelial electrochemical gradient for H+ significantly from 30 +/- 4 mV to 14 +/- 3 mV. The results suggest: In the diluting segment of the frog kidney a Na+-dependent rheogenic HCO3- transport system exists across the peritubular cell membrane. This rheogenic peritubular Na+/HCO3- cotransporter cooperates with a Na+/H+ exchanger in the luminal membrane, thus driving HCO3- reabsorption. Reabsorption of HCO3- and secretion of H+ depend upon the presence of carbonic anhydrase.

The hypothesis was tested whether amiloride and/or an acute acid load influence Cl- transport in the diluting segment of the isolated-perfused kidney of the K+ adapted frog (rana pipiens). Transepithelial resistance (luminal cable analysis) and Cl- net flux (Cl- sensitive microelectrodes) were evaluated at various concentrations of amiloride, at high pCO2 or low HCO-3 in the kidney perfusate. Amiloride or an acute acid load increase transepithelial resistance. The resistance-change at given concentrations of amiloride is markedly enhanced under static head conditions, i.e. at low luminal NaCl concentrations. Amiloride or acidosis (high pCO2) reduce Cl- net reabsorption; combination of both potentiates this inhibitory effect. We conclude: an acute acid load acidifies the cell cytosol. This effect is aggravated dramatically after amiloride-induced inhibition of the luminal Na+/H+ exchanger. The luminal pH-sensitive K+ conductance decreases. This results in a depolarization of the cell membranes. Consequently, the peritubular electrochemical driving force for the exit step of Cl- (from cell to blood) dissipates. Therefore, Cl- net reabsorption is blunted.

The dependence of intracellular pH (pHi) and transepithelial H+ secretion on the cell membrane potential (Vm) was tested applying pH-sensitive and conventional microelectrodes in giant cells fused from single epithelial cells of the diluting segment and in intact tubules of the frog kidney. An increase of extracellular K+ concentration from 3 to 15 mmol/l decreased Vm from -49 +/- 4 to -29 +/- 1 mV while pHi increased from 7.44 +/- 0.04 to 7.61 +/- 0.06. Addition of 1 mmol/l Ba2+ depolarized Vm from -45 +/- 3 to -32 +/- 2 mV, paralleled by an increase of pHi from 7.46 +/- 0.04 to 7.58 +/- 0.03. Application of 0.05 mmol/l furosemide hyperpolarized Vm from -48 +/- 3 to -53 +/- 3 mV and decreased pHi from 7.47 +/- 0.05 to 7.42 +/- 0.05. In the intact diluting segment of the isolated-perfused frog kidney an increase of peritubular K+ concentration from 3 to 15 mmol/l increased the luminal pH from 7.23 +/- 0.08 to 7.41 +/- 0.08. Addition of Ba2+ to the peritubular perfusate also increased luminal pH from 7.35 +/- 0.07 to 7.46 +/- 0.07. Addition of furosemide decreased luminal pH from 7.32 +/- 0.03 to 7.24 +/- 0.05. We conclude: cell depolarization reduces the driving force for the rheogenic HCO3- exit step across the basolateral cell membrane. HCO3- accumulates in the cytoplasm and pHi increases. An alkaline pHi inactivates the luminal Na+/H+ exchanger. This diminishes transepithelial H+ secretion. Cell hyperpolarization leads to the opposite phenomenon. Thus, pHi serves as signal transducer between cell voltage and Na+/H+ exchange.

Increased chronic intake of K+ induces H+ and K+ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed) Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pHlu) was measured continuously with pH-sensitive microelectrodes while aldosterone (3 X 10(-7) to 3 X 10(-6) mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (Vte) from 8.5 +/- 1.1 mV to 4.0 +/- 0.6 mV pHlu dropped from 7.73 +/- 0.02 to a new steady-state value of 7.17 +/- 0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10(-3) mol/l amiloride reversed luminal acidification to a pHlu of 7.68 +/- 0.04; at the same time Vte recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone--after interaction with cytoplasmic receptors--activates the luminal, amiloride-inhibitable Na+/H+ exchanger. This mechanism could explain enhanced H+ secretion found in the K+ adapted animal.