Chloride transport in normal and cystic fibrosis epithelial cells

The experimental work described in this thesis was aimed at a better understanding of the pathogenesis of CF with the emphasis on electrophysiological studies on Cl" transport. Chloride transport was studied with Ussing chamber, isotopic ('25I")-efflux and fluorescent (SPQ) measurements as well as with the patch-damp technique. In search for a suitable cell culturing model, we studied C!" transport in CF keratinocytes, which are easy to obtain and would provide us with sufficient cell material (Chapter 3). However, the use of keratinocytes appeared to be limited to primary cell cultures which can only be passaged for maximal 10 times, necessatating the use of many different and often heterogeneous N and CF cell lines necessary. The availibility of a continuously growing cell line with CF genotype and phenotype would provide a more homogeneous model for biochemical, electrophysiological and genetic studies. We have therefore immortalized a CF nasal polyp cell line with a hybrid SV40/ Ad12 virus, and have characterized this cell line biochemically and electrophysiologically as discussed in Chapter 4. A continuously growing homogeneous cell line selected for its chloride secreting characteristics would also he of much interest for CF research. The subclone 19A of the human colon carcinoma cell line HI29 is such a cell line, easy to maintain in culture without the multiple addition;; to the medium required for culturing keratinocytes and primary nasal polyp cells and without the use of feeder cells. These colonocytes can be coru;idered as a model for the study of Cl· transport in normal cells. In Chapter 5 we describe the patchclamp experiments with HI29.ci19A cells performed to characterize the cAMPdependent regulation of the outwardly rectifYing chloride channel which was thought to be defectively regulated in CF. Apart from cAMP regulation of chloride channels, we also found that G-proteiru; are involved in the regulation of a different type of chloride channel in intestinal membranes. Chapter 6 describes the vesicle and patch-damp experiments leading to the identification of a novel potential signal transduction pathway for chloride transport in intestinal cells. The homology between CFIR and different types of pump proteins like MDR led us to verify the hypothesis that CFIR could function as a pump with substrates exerting secondary effects on chloride channels. In Chapter 7 MDR-substrates and -blockers were tested on epithelial chloride traru;port and on the outwardly rectifYing chloride channel in particular

Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A

Cell-attached patch-clamp studies with the human colon carcinoma HT-29cl.19A cells revealed a small chloride channel with a unitary conductance of 6.5 pS at 70 mV and 4.6 pS at -70 mV clamp potential after cAMP was increased by activation of adenylyl cyclase by forskolin. Usually channels inactivated upon patch excision, but in a few excised patches the channels stayed active and displayed a linear I/V relation in symmetrical (150 mmol/l) chloride solutions with a conductance of 7.5 pS. A 16-fold increase in channel incidence was observed when forskolin and phorbol 12,13-dibutyrate (PDB) were present together. The open probability was voltage-independent and was not different in the presence of forskolin plus PDB or with forskolin alone. The conductance sequence of the channel as deduced from outward currents carried by five different anions including chloride was: Cl->Br->NO3 ->gluconate > I-. The permeability sequence deduced from the reversal potentials was NO3 -≥Br->Cl->I->gluconate. With iodide in the pipette the conductance decreased strongly. Moreover, the inward current was reduced by 61%, indicating a strong inhibition of the chloride efflux by iodide. Similarly, the forskolin-induced increase of the short-circuit current (Isc) in confluent filter-grown monolayers was strongly reduced by iodide in the apical perfusate. Iodide also increased the fractional resistance of the apical membrane and repolarized the membrane potential, indicating an inhibitory action on the forskolin-induced increase of the apical chloride conductance. The PDB-induced Isc was also reduced by iodide, suggesting that the same chloride conductance is involved in the forskolin and in the PDB response. The results suggest that forskolin via cAMP-dependent protein kinase and PDB via protein kinase C regulate the same non-rectifying small-conductance chloride channels in the HT-29cl.19A cells

Regulation of chloride transport in cultured normal and cystic fibrobis keratinocytes

Abstract Cultured normal (N) and cystic fibrosis (CF) keratinocytes were evaluated for their Cl−-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl− channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl− channel was found with a similar incidence in N and CF apical keratininocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successfull in either N or CF keratinocytes. Forskolin was not able to activate Cl− channels in N and CF cell-attached patches. The Ca2+-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl− channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl− transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl− and 125I−. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl−-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways

Improving diagnostics and quality of life in children with peanut allergy and other atopic diseases

Peanut allergy disrupts the daily lives of children and their parents. Families with a peanut allergic child often live with increased stress, anxiety and constant vigilance about food consumption. The aim of this thesis was to improve the diagnostics and quality of life in children with peanut allergy and their parents. Peanut allergy diagnostics can be improved using a blood test (sIgE to peanut protein Ara h 2). In current clinical practice, patients with a suspected peanut allergy undergo a two-day food challenge to confirm or exclude a peanut allergy which is burdensome and costly. Results from this thesis indicate that children with a very low blood test result (20% of all children) can safely introduce peanut at home, without food-challenge in hospital. In children with a high blood test result (35% of all children), the two-day food challenge can be replaced by a one-day food challenge to confirm peanut allergy. This thesis indicates that quality of life improves after a food challenge. Anxiety decreases in parents of children with a suspected peanut allergy with a very low blood test result. These results indicate that families benefit form clear peanut allergy diagnostics. We recommend to use the blood test as described in this thesis in all children with a suspected peanut allergy in the Netherlands. This could lead to a cost reduction of up to 1.5 million euros per year

Improving diagnostics and quality of life in children with peanut allergy and other atopic diseases

Peanut allergy disrupts the daily lives of children and their parents. Families with a peanut allergic child often live with increased stress, anxiety and constant vigilance about food consumption. The aim of this thesis was to improve the diagnostics and quality of life in children with peanut allergy and their parents. Peanut allergy diagnostics can be improved using a blood test (sIgE to peanut protein Ara h 2). In current clinical practice, patients with a suspected peanut allergy undergo a two-day food challenge to confirm or exclude a peanut allergy which is burdensome and costly. Results from this thesis indicate that children with a very low blood test result (20% of all children) can safely introduce peanut at home, without food-challenge in hospital. In children with a high blood test result (35% of all children), the two-day food challenge can be replaced by a one-day food challenge to confirm peanut allergy. This thesis indicates that quality of life improves after a food challenge. Anxiety decreases in parents of children with a suspected peanut allergy with a very low blood test result. These results indicate that families benefit form clear peanut allergy diagnostics. We recommend to use the blood test as described in this thesis in all children with a suspected peanut allergy in the Netherlands. This could lead to a cost reduction of up to 1.5 million euros per year