41
Biochimica et Biophysica Acta, 1139 (1992) 41-48
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBADIS 61151
Chloride transport in control and cystic fibrosis human skin fibroblast membrane vesicles Monique Vasseur, Michele Caiizac, Isabelle Garcia l and Francisco Alvarado Centre de Recherche sur l'Endocrinologie Mol~eulaire et le D~veloppement, Centre National de la Recherche Scientifique, Meudon (France)
(Received 18 April 1991) (Revised manuscript received 18 December 1991)
Key words: Human skin fibroblast; Cystic fibrosis; Membrane vesicle; Chloride tranport Plasma membrane vesicles were isolated from either cystic fibrosis (CF) or non-CF cultured fibroblasts derived from skin biopsies of either foetus, child or adolescent human donors. The total membrane yield was essentially identical for either CF or control membranes. By using a rapid filtration technique, 36C1 uptake by these vesicles was quantitated in the absence and presence of alkali-metal ion-, electrical- a n d / o r pH gradients. In the absence of a pH gradient (pHou t = PHin = 7.5), C1 uptake took place downhill in both cases. Either cis K +, cis Na + or an equimolar mixture of cis Na + plus K + caused CI uptake activation. In the presence of an alkaline-inside pH gradient (PHout/PHin = 5.5/7.5), C1 uptake exhibited an apparent overshoot independently of the presence or absence of any metal-ion gradient. The observed potassium-, sodium- and proton-dependent C1 influx rates were all unaffected by voltage clamping, indicating the existence in these vesicles of electroneutral symport systems of the type Cl / H +, Cl / K + a n d / o r CI / N a + ; but not 2 C I - / N a + / K +. In the presence of an inward-directed K + gradient, valinomycin further increased CI uptake, both in the presence and in the absence of a pH gradient, indicating the presence of a rheogenic CI uniport. In absolute quantitative terms, the two different modes (rheogenic and electroneutral) of C1 transport evinced in these vesicles were about 45% lower in CF than in control skin fibroblasts. However, qualitatively, there was no difference between normal and CF cells. The evidence obtained indicates that the CF defect, which is expressed in fibroblast plasma membranes, does not affect specifically either the rheogenic or the electroneutral Cl transport systems. Rather, the CF cells appear to give a smaller yield of closed, functional vesicles, reflected by a significantly smaller apparent intravesicular volume. Because it also affects the transport of D-glucose and L-alanine, this anomaly could be the consequence of a generalized membrane defect characterizing CF fibroblasts.
Introduction R e c e n t l y , the g e n e t i c d e f e c t in cystic fibrosis ( C F ) has b e e n t r a c e d to t h e d e l e t i o n o f a t r i p l e t c o d i n g for p h e n y l a l a n i n e ( m u t a t i o n A F - 5 0 8 ) in a g e n e whose p r o d u c t is the s o - c a l l e d C F t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r ( C F T R ) p r o t e i n [1]. H o w e v e r , the prim a r y lesion in C F r e m a i n s u n e x p l a i n e d , even if m u c h p r o g r e s s has b e e n m a d e recently, i n d i c a t i n g t h a t this
1 Present address: Laboratoire de Chimie Clinique, H6pital Sainte Eugenie, Saint Genis Laval, France. Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone. The suffixes out and in indicate respectively the extra- and the intravesicular media. CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator. Correspondence: F. Alvarado, Centre de Recherche sur l'Endocrinologie Mol6culaire et le D6veloppement du C.N.R.S., 9, rue Hetzel, 92190 Meudon, France.
d i s e a s e involves a m o l e c u l a r d e f e c t in m e m b r a n e ion t r a n s p o r t . M o r e precisely, r e c e n t r e s e a r c h , b a s e d to a large extent on using the p a t c h - c l a m p t e c h n i q u e , has singled out as a key factor in C F a dysfunction in the r h e o g e n i c t r a n s f e r o f chloride, t h e so-called c h l o r i d e c h a n n e l , a n d / o r its r e g u l a t i o n (for r e c e n t reviews, see Refs. 2 a n d 3). T h e C1 c h a n n e l dysfunction has b e e n d e s c r i b e d for a variety of cells, b o t h e p i t h e l i a l ( t r a c h e a l , nasal polyps, Refs. 4 - 6 ) a n d n o n - e p i t h e l i a l (fibroblasts, lymhocytes, Refs. 7 - 1 0 ) . H o w e v e r , several q u e s t i o n s r e m a i n u n a n swered. First, it s e e m s unlikely t h a t the C1 c h a n n e l s d e t e c t e d in the a b o v e - m e n t i o n e d v a r i e t y o f cells constitute o n e single m o l e c u l a r entity, which p e r m i t s raising the question: if at least s o m e o f t h e s e c h a n n e l s a r e m o l e c u l a r l y d i f f e r e n t from o n e a n o t h e r , how is it t h a t they a r e all a f f e c t e d as a c o n s e q u e n c e o f a single-point m u t a t i o n ? S e c o n d , is the c h l o r i d e c h a n n e l the only m e m b r a n e t r a n s p o r t system a f f e c t e d in C F ? T h e p a t c h - c l a m p t e c h n i q u e m e a s u r e s only fluxes t h a t a r e
42 rheogenic, meaning that a defect possibly involving some other, elcctroneutraI mechanism, would go unnoticed. By using a rapid filtration technique, we showed previously that intestinal CI transport across brush border m e m b r a n e vesicles from guinea-pig ileum involves mainly an electroneutral CI / H + symport (CI-/OH antiport), but exhibits little rheogenic CI u n i p o r t and n e i t h e r CI / N a +, CI / K ' o r 2 C I - / N a + / K + symport nor obligatory C1 /C1 or C I - / H C O 3 antiport activities [11]. Because there is little information on the possible contribution of electroneutral mechanisms to total C1 fluxes in both CF and control cells, in the present work we set out to investigate, by applying the same approach, C1 transport across m e m b r a n e vesicles isolated from cultured human skin fibroblasts. This material can be obtained readily, and, for reasons still not understood, seems to be affected in CF [7-9], contrasting with the fact that the C F T R gene product appears not to be detectable in fibroblasts [1]. Consequently, we have quantitated various modes of m e m b r a n e C1 transport in human skin fibroblasts, our aim being to ascertain whether or not both electrogenic (rheogenic) and electroneutral CI transport systems exist and are specifically affected in CF. Materials and Methods
Materials. H36Cl (0.4 m C i / m m o l , A m e r s h a m ) was neutralized with Tris base before use. Valinomycin, CCCP (carbonyl cyanide rn-chlorophenylhydrazone) and other chemicals of the highest purity were purchased from Sigma. Buffer composition. For all experiments, unless stated otherwise, both the extra- and the intravesicular media contained the same buffer consisting of 20 mM H e p e s / 4 0 mM citric acid, adjusted to the desired pH with Tris base. The ionic strength of the various buffers was maintained as nearly constant as possible by adding as needed the appropriate amount of 'inert' base a n d / o r acid, respectively, Tris base and gluconic acid. All media were adjusted with sorbitol to maintain an osmolarity ratio ((out/in) of 650/550 mOsmol per liter. Valinomycin and CCCP, dissolved in ethanol, were allowed to evaporate to dryness before mixing with the m e m b r a n e preparation, which took place at least 15 min prior to assaying for chloride transport [11]. Each ionophore was used in the concentration of 1 0 / x g / m g of m e m b r a n e protein [11]. Human skin fibroblasts in culture. CF and control fibroblast cultures were established from skin biopsies of either foetus, child (1-20 months) or adolescent (15-20 years) humans. Two cell lots per condition were used, except for child fibroblasts where only one con-
trol and one CF lot were available. The biopsm~ ~vcr,: obtained at the HOpital Saintc ['Ltlgcnie. Saint
Biochimica et Biophysica Acta, 1139 (1992) 41-48
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBADIS 61151
Chloride transport in control and cystic fibrosis human skin fibroblast membrane vesicles Monique Vasseur, Michele Caiizac, Isabelle Garcia l and Francisco Alvarado Centre de Recherche sur l'Endocrinologie Mol~eulaire et le D~veloppement, Centre National de la Recherche Scientifique, Meudon (France)
(Received 18 April 1991) (Revised manuscript received 18 December 1991)
Key words: Human skin fibroblast; Cystic fibrosis; Membrane vesicle; Chloride tranport Plasma membrane vesicles were isolated from either cystic fibrosis (CF) or non-CF cultured fibroblasts derived from skin biopsies of either foetus, child or adolescent human donors. The total membrane yield was essentially identical for either CF or control membranes. By using a rapid filtration technique, 36C1 uptake by these vesicles was quantitated in the absence and presence of alkali-metal ion-, electrical- a n d / o r pH gradients. In the absence of a pH gradient (pHou t = PHin = 7.5), C1 uptake took place downhill in both cases. Either cis K +, cis Na + or an equimolar mixture of cis Na + plus K + caused CI uptake activation. In the presence of an alkaline-inside pH gradient (PHout/PHin = 5.5/7.5), C1 uptake exhibited an apparent overshoot independently of the presence or absence of any metal-ion gradient. The observed potassium-, sodium- and proton-dependent C1 influx rates were all unaffected by voltage clamping, indicating the existence in these vesicles of electroneutral symport systems of the type Cl / H +, Cl / K + a n d / o r CI / N a + ; but not 2 C I - / N a + / K +. In the presence of an inward-directed K + gradient, valinomycin further increased CI uptake, both in the presence and in the absence of a pH gradient, indicating the presence of a rheogenic CI uniport. In absolute quantitative terms, the two different modes (rheogenic and electroneutral) of C1 transport evinced in these vesicles were about 45% lower in CF than in control skin fibroblasts. However, qualitatively, there was no difference between normal and CF cells. The evidence obtained indicates that the CF defect, which is expressed in fibroblast plasma membranes, does not affect specifically either the rheogenic or the electroneutral Cl transport systems. Rather, the CF cells appear to give a smaller yield of closed, functional vesicles, reflected by a significantly smaller apparent intravesicular volume. Because it also affects the transport of D-glucose and L-alanine, this anomaly could be the consequence of a generalized membrane defect characterizing CF fibroblasts.
Introduction R e c e n t l y , the g e n e t i c d e f e c t in cystic fibrosis ( C F ) has b e e n t r a c e d to t h e d e l e t i o n o f a t r i p l e t c o d i n g for p h e n y l a l a n i n e ( m u t a t i o n A F - 5 0 8 ) in a g e n e whose p r o d u c t is the s o - c a l l e d C F t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r ( C F T R ) p r o t e i n [1]. H o w e v e r , the prim a r y lesion in C F r e m a i n s u n e x p l a i n e d , even if m u c h p r o g r e s s has b e e n m a d e recently, i n d i c a t i n g t h a t this
1 Present address: Laboratoire de Chimie Clinique, H6pital Sainte Eugenie, Saint Genis Laval, France. Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone. The suffixes out and in indicate respectively the extra- and the intravesicular media. CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator. Correspondence: F. Alvarado, Centre de Recherche sur l'Endocrinologie Mol6culaire et le D6veloppement du C.N.R.S., 9, rue Hetzel, 92190 Meudon, France.
d i s e a s e involves a m o l e c u l a r d e f e c t in m e m b r a n e ion t r a n s p o r t . M o r e precisely, r e c e n t r e s e a r c h , b a s e d to a large extent on using the p a t c h - c l a m p t e c h n i q u e , has singled out as a key factor in C F a dysfunction in the r h e o g e n i c t r a n s f e r o f chloride, t h e so-called c h l o r i d e c h a n n e l , a n d / o r its r e g u l a t i o n (for r e c e n t reviews, see Refs. 2 a n d 3). T h e C1 c h a n n e l dysfunction has b e e n d e s c r i b e d for a variety of cells, b o t h e p i t h e l i a l ( t r a c h e a l , nasal polyps, Refs. 4 - 6 ) a n d n o n - e p i t h e l i a l (fibroblasts, lymhocytes, Refs. 7 - 1 0 ) . H o w e v e r , several q u e s t i o n s r e m a i n u n a n swered. First, it s e e m s unlikely t h a t the C1 c h a n n e l s d e t e c t e d in the a b o v e - m e n t i o n e d v a r i e t y o f cells constitute o n e single m o l e c u l a r entity, which p e r m i t s raising the question: if at least s o m e o f t h e s e c h a n n e l s a r e m o l e c u l a r l y d i f f e r e n t from o n e a n o t h e r , how is it t h a t they a r e all a f f e c t e d as a c o n s e q u e n c e o f a single-point m u t a t i o n ? S e c o n d , is the c h l o r i d e c h a n n e l the only m e m b r a n e t r a n s p o r t system a f f e c t e d in C F ? T h e p a t c h - c l a m p t e c h n i q u e m e a s u r e s only fluxes t h a t a r e
42 rheogenic, meaning that a defect possibly involving some other, elcctroneutraI mechanism, would go unnoticed. By using a rapid filtration technique, we showed previously that intestinal CI transport across brush border m e m b r a n e vesicles from guinea-pig ileum involves mainly an electroneutral CI / H + symport (CI-/OH antiport), but exhibits little rheogenic CI u n i p o r t and n e i t h e r CI / N a +, CI / K ' o r 2 C I - / N a + / K + symport nor obligatory C1 /C1 or C I - / H C O 3 antiport activities [11]. Because there is little information on the possible contribution of electroneutral mechanisms to total C1 fluxes in both CF and control cells, in the present work we set out to investigate, by applying the same approach, C1 transport across m e m b r a n e vesicles isolated from cultured human skin fibroblasts. This material can be obtained readily, and, for reasons still not understood, seems to be affected in CF [7-9], contrasting with the fact that the C F T R gene product appears not to be detectable in fibroblasts [1]. Consequently, we have quantitated various modes of m e m b r a n e C1 transport in human skin fibroblasts, our aim being to ascertain whether or not both electrogenic (rheogenic) and electroneutral CI transport systems exist and are specifically affected in CF. Materials and Methods
Materials. H36Cl (0.4 m C i / m m o l , A m e r s h a m ) was neutralized with Tris base before use. Valinomycin, CCCP (carbonyl cyanide rn-chlorophenylhydrazone) and other chemicals of the highest purity were purchased from Sigma. Buffer composition. For all experiments, unless stated otherwise, both the extra- and the intravesicular media contained the same buffer consisting of 20 mM H e p e s / 4 0 mM citric acid, adjusted to the desired pH with Tris base. The ionic strength of the various buffers was maintained as nearly constant as possible by adding as needed the appropriate amount of 'inert' base a n d / o r acid, respectively, Tris base and gluconic acid. All media were adjusted with sorbitol to maintain an osmolarity ratio ((out/in) of 650/550 mOsmol per liter. Valinomycin and CCCP, dissolved in ethanol, were allowed to evaporate to dryness before mixing with the m e m b r a n e preparation, which took place at least 15 min prior to assaying for chloride transport [11]. Each ionophore was used in the concentration of 1 0 / x g / m g of m e m b r a n e protein [11]. Human skin fibroblasts in culture. CF and control fibroblast cultures were established from skin biopsies of either foetus, child (1-20 months) or adolescent (15-20 years) humans. Two cell lots per condition were used, except for child fibroblasts where only one con-
trol and one CF lot were available. The biopsm~ ~vcr,: obtained at the HOpital Saintc ['Ltlgcnie. Saint
