Volume 119, Issue 1, Pages 32-40 (July 2000) Residual chloride secretion in intestinal tissue of ΔF508 homozygous twins and siblings with cystic fibrosis  Inez Bronsveld, Frauke Mekus, Jan Bijman, Manfred Ballmann, Joachim Greipel, Joachim Hundrieser, Dicky J.J. Halley, Ulrike Laabs, Roger Busche, Hugo R. De Jonge, Burkhard Tümmler, Henk J. Veeze  Gastroenterology  Volume 119, Issue 1, Pages 32-40 (July 2000) DOI: 10.1053/gast.2000.8524 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Protocol of the ICM measurement. (A) Healthy control, with a large increase in Isc upon addition of carbachol and histamine representing apical Cl− secretion. (B) Typical CF patient in whom carbachol, 8-bromo-cAMP + forskolin, and histamine did not evoke Cl− exchange across the apical membrane. The reversed response implies apical K+ efflux. Note not only the difference in direction of the Isc response compared with control, but also the difference in scale. (C) CF patient with residual Cl− transport that is not influenced by the presence of DIDS: carbachol, 8-bromo-cAMP + forskolin, and histamine evoke residual Cl− secretion. (D) CF patient with DIDS-sensitive residual Cl− secretion, which is characterized by an increase in Isc in response to carbachol and 8-bromo-cAMP + forskolin but a decrease in Isc upon stimulation with histamine in the presence of DIDS. The tracings are original recordings of individuals who express an ICM pattern representative of the subgroup of patients all showing that specific ICM response pattern. Gastroenterology 2000 119, 32-40DOI: (10.1053/gast.2000.8524) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Quantitative interpretation of the Cl−-secretory responses to carbachol and histamine. (A) Sketch of a Cl−-secretory response. A baseline is assigned to the response (dotted line) on the basis of 2 linear segments on the recording, 1 before and 1 after the secretory response (segments between crosses). With respect to this baseline, the heights of the reversed and residual responses are determined (between dashed lines). The amount of residual Cl− conductance (ΔIscCl) is calculated as a percentage of the total response by dividing the height of the peak in the chloride-secretory direction by the height of the total response (PUH%). (B) On the basis of the residual Cl− secretion expressed in PUH%, carbachol and histamine responses are categorized into 5 classes. Experimental examples of tracings are shown for each class. For comparison, the carbachol and histamine responses of the CF patients presented in Figure 1 are thus classified as: Figure 1B, carbachol class V, histamine class V; Figure 1C, carbachol class II, histamine class II; Figure 1D, carbachol class I, histamine class V. Gastroenterology 2000 119, 32-40DOI: (10.1053/gast.2000.8524) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Quantitative interpretation of the Cl−-secretory responses to carbachol and histamine. (A) Sketch of a Cl−-secretory response. A baseline is assigned to the response (dotted line) on the basis of 2 linear segments on the recording, 1 before and 1 after the secretory response (segments between crosses). With respect to this baseline, the heights of the reversed and residual responses are determined (between dashed lines). The amount of residual Cl− conductance (ΔIscCl) is calculated as a percentage of the total response by dividing the height of the peak in the chloride-secretory direction by the height of the total response (PUH%). (B) On the basis of the residual Cl− secretion expressed in PUH%, carbachol and histamine responses are categorized into 5 classes. Experimental examples of tracings are shown for each class. For comparison, the carbachol and histamine responses of the CF patients presented in Figure 1 are thus classified as: Figure 1B, carbachol class V, histamine class V; Figure 1C, carbachol class II, histamine class II; Figure 1D, carbachol class I, histamine class V. Gastroenterology 2000 119, 32-40DOI: (10.1053/gast.2000.8524) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Classification of Cl−-secretory responses to carbachol and histamine according to PUH% for 55 ΔF508 homozygous CF patients. The carbachol (vertical) and histamine (horizontal) responses are categorized for 41 CF patients belonging to dizygous pairs (●) and for 14 patients belonging to monozygous twin pairs (♦). The influence of DIDS on chloride secretion is indicated as follows: upper left black-bordered square, patients with DIDS-insensitive residual Cl− secretion, interpreted as the presence of residual CFTR activity; dark gray areas, patients expressing DIDS-sensitive residual Cl− secretion, pointing to the presence of alternative Cl− conductances; light gray areas, patients who show residual Cl− secretion in the presence but not in the absence of DIDS. For clarification, the ICM patterns of the 3 CF patients in Figure 1 are assigned to their corresponding carbachol and histamine classes by annotation of 1B, 1C, and 1D, referring to the ICM patterns of Figure 1B, C, and D. Gastroenterology 2000 119, 32-40DOI: (10.1053/gast.2000.8524) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Concordance of the Cl−-secretory responses to carbachol and histamine within patient pairs. Classification of (A) carbachol responses of sibling A (vertical) vs. sibling B (horizontal) for 29 sibling and dizygous twin pairs (●) and 9 monozygous twin pairs (♦). (B) Classification of histamine responses for 18 dizygous pairs (○) and 5 monozygous pairs (♢). The gray areas contain all pairs that are concordant in their Cl−-secretory responses, i.e., siblings A and B do not differ more than 2 classes in their carbachol and histamine responses. Gastroenterology 2000 119, 32-40DOI: (10.1053/gast.2000.8524) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Concordance of the Cl−-secretory responses to carbachol and histamine within patient pairs. Classification of (A) carbachol responses of sibling A (vertical) vs. sibling B (horizontal) for 29 sibling and dizygous twin pairs (●) and 9 monozygous twin pairs (♦). (B) Classification of histamine responses for 18 dizygous pairs (○) and 5 monozygous pairs (♢). The gray areas contain all pairs that are concordant in their Cl−-secretory responses, i.e., siblings A and B do not differ more than 2 classes in their carbachol and histamine responses. Gastroenterology 2000 119, 32-40DOI: (10.1053/gast.2000.8524) Copyright © 2000 American Gastroenterological Association Terms and Conditions