Cystic fibrosis and phosphatidylcholine biosynthesis

Clinica Chimica Acta 230 (1994) 109- 116

ELSEVIER

Cystic fibrosis and phosphatidylcholine

biosynthesis

Marta M. Ulanea, Jean DeB. Butlerb, Alessandro Perib, Lucia Mieleb, Rodney E. Ulanea, Van S. Hubbard*a, aPediatric Metabolism Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Westwood Building, Room 3A18, Bethesda, IUD 20892 USA bHuman Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

Received 29 September 1993;revision received 7 April 1994;accepted I I April 1994

Abstract

The cystic fibrosis (CF) gene defect may be associated with a defect in membrane recycling. We have investigated the metabolism of the main constituent of plasma membrane, phosphatidylcholine (PC). In this study of platelets and fibroblasts, we show an increased uptake of choline into PC of CF cells as compared with normal cells. No accumulation of PC was seen. Other patients with respiratory disease (not CF) showed normal rates of incorporation of choline into platelet PC. Platelets from heterozygote individuals showed intermediate turnover rates of choline incorporation into PC. The increase in choline incorporation into PC in CF platelets was not due to modified or increased sensitivity to either CAMP or prostaglandin E,. The total amount and the proportions of the major phospholipids in platelets of control and CF individuals were identical. These findings indicate an increased turnover rate of this phospholipid in CF cells rather than an increased net synthesis. Keywords:

Cystic fibrosis; Phosphatidylcholine;

Choline; Fatty acids; Membranes

1. Introduction Cystic fibrosis (CF) is an autosomal recessive genetic disorder. The gene responsible for this disease has recently been shown to code for a regulated membrane Cl- ion channel [l-5]. CF is clinically characterized by chronic pulmonary disease, l Corresponding author, Nutritional Sciences Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Westwood Building, Room 3A18, Bethesda, MD 20892, USA.

0009-8981/94607.00 Elsevier Science B.V. SSDI 0009-8981(94)05933-J

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pancreatic insufficiency and elevated concentrations of electrolytes in the sweat [6]. At the cellular level, there is a malfunction in Cl- and Na+ ion movements in the sweat glands and epithelial cells of CF patients [7-lo]. It has also been demonstrated that tissues from CF patients contain abnormally high levels of mucus glycoproteins. The cystic fibrosis transmembrane regulator (CFTR) gene product has been found to be present and active also in endosomes [l l] and has been suggested to be involved in the regulation of endosomal pH [ 121. More recently, it has been suggested that CFTR participates in the regulation of membrane recycling through endo- and exocytosis [ 131.Thus it is possible that membrane phospholipid metabolism may be affected by the CFTR defect in CF cells. Supporting this proposition is the finding that the fatty acid composition of the phospholipid fractions of red blood cell membranes of CF patients differs from that found in healthy individuals [14]. The turnover rates of palmitic and linoleic acids in these phospholipids were also found to be higher in CF patients (151. Furthermore, the fatty acid compositions of other tissues, including plasma [ 161, adipose tissue [ 171, bronchial mucus [ 18,191 and platelets [20,21] have been found to differ in CF patients when compared with healthy individuals [22]. Altered regulation of arachidonic acid release [23] and increased breakdown of phosphatidylinositol in erythrocyte membranes of CF patients has also been reported [24]. In this study, the biosynthesis of PC, the predominant phospholipid component of the cell membrane, was investigated in blood platelets and skin tibroblasts of CF and healthy individuals. 2. Materials and methods 2.1. Patients Thirteen patients, 5 obligate heterozygotes, 17 normal subjects and three patient controls were studied. All CF patients had a medical history and clinical evaluation indicative of the diagnosis of CF and a positive sweat test. Obligate heterozygotes were parents of a CF patient. Normal subjects were volunteers without history or symptoms of respiratory or gastrointestinal disease. Patient controls were subjects with nor&F, chronic respiratory disease. Informed consent was obtained from all patients. 2.2. Platelet preparation Blood was drawn into plastic tubes containing 0.38% sodium citrate (final concentration). Platelet-rich plasma was prepared by centrifugation at 300 x g for 20 min at 4°C. The platelet pellet was resuspended in Hepes-buffered Hank’s Balanced Salt Solution, without calcium or magnesium (HBSS) and containing 1% EDTA, and washed twice at 1800 x g for 10 min at 4OC. Subsequently, platelets were washed in HBSS with 0.5% EDTA at 1200 x g for 10 min at 4°C. The final pellet was resuspendsed in HBSS containing 0.1% EDTA. Platelet protein was determined by the method of Lowry et al., using bovine albumin as protein standard [25]. 2.3. Measurement of phosphatidylcholine synthesis The incubation mixture contained 0.8 mg of platelet protein in HBSS with 0.1%

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EDTA, 1 mM CaCl*, 1 mM MgCl* and 1 &i of U-[‘4C]choline (sa., 1 &i/pmol). In some experiments, 37.5 mM dibutyryl CAMP and 0.1 mM prostaglandin E2 were also added to the incubation solutions. Incubations were stopped at 60 and 90 min by the addition of 10 PM choline chloride in HBSS containing 1% EDTA and centrifuging at 1800 x g for 10 min at 4°C. The platelet pellet was resuspended in HBSS containing 1% EDTA. Lipids were extracted with a solution of chloroform and methanol (1: 1, v/v) using 4 ml organic solvent to 2 ml of platelet resuspension and the mixture vortexed and centrifuged at 1800 x g for 20 min at 4°C. The aqueous phase was discarded, the organic phase transferred to scintillation vials and counted for i4C radioactivity. 2.4. Phospholipid extraction and composition Platelets were washed in HBSS containing 1% EDTA, resuspended and mixed in 2.8 ml of the same buffer plus 2.0 ml of chloroform and 2.0 ml of methanol and the aqueous phase discarded. The organic phase was evaporated, then redissolved in a chloroform/methanol mixture (2:1, v/v) and applied to Baker Silica Gel G TLC plates. The plates were developed in petroleum ether/chloroform/acetic acid/boric acid/methanol (30 ml:40 ml:10 ml: 1.8 g:20 ml). Ten micrograms each of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), lysophosphatidylcholine (LPC) and phosphatidylethanolamine (PE) were applied to the plates as standards and visualized with iodine vapors. Appropriate areas of the developed plates were scraped for counting radioactivity and for the measurement of organic phosphate by the method of Bartlett [26]. 2.5. Fibroblasts Fibroblasts were obtained from skin biopsies of CF patients and normal volunteers after obtaining written consent. CF diagnosis was confirmed by sweat chloride determination [6]. Cells were maintained in tissue culture using standard procedures and incubated in Eagle’s Minimal Essential Medium (EMEM) containing 10% fetal bovine serum and 2 mM glutamine. Cells were incubated at 37°C in a 5% CO2 atmosphere. 2.4. PCR amplification Genomic DNAs extracted from human skin libroblasts were subjected to PCR using oligonucleotide primers derived from the sequence of CFTR cDNA. The sequence of the sense primer (CFD-L) was: 5 ‘-GGAGAACTGGAGCCTTCAGA-3 ’ (T,,, 69”C, nucleotides 1549-1568 of CFTR cDNA). The sequence of the antisense primer (CFD-R) was: 5 ‘-AGTTGGCATGCTTTGATGAC-3 ’ (T,,, 69”C, nucleotides 1709-1690). PCR amplification was performed as follows: 2 min at 95°C (1 cycle); 1 min at 95°C plus 1 min at 64°C plus 1 min at 72°C (40 cycles); 10 min at 72°C (1 cycle). 2.7. Detection and characterization of PCR products The PCR products were electrophoresed in agarose gels and the presence of CFTR-specific bands was confirmed by Southern blotting using two different probes from exon 10 of the CFTR gene. The sequence of the wild type probe (CFP-N) was:

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5 ‘-ATATCATCTMGGTGTTTCC-3 ’ (T, 62’C) [l-3], corresponding to nucleotides 1646-1665 of CFTR cDNA. The sequence of the mutant probe (CFP-AF) was: 5 ‘-AAATATCATTGGTGTTTCCTA-3 ’ T,,, 62°C corresponding to positions 1644-1667 of the wild type gene sequence. The probes were labeled at the 3 ’ end with digoxigenin-11-ddUTP using the Genius 5 kit (Boehringer Mannheim Corp., Indianapolis, IN). The hybridization was carried out for 4 h at 57°C. Post-hybridization washes were performed as follows: 2 x 5 min at 37°C in 2 x SSC, 0.1% SDS; 1 x 5 min at 57°C in 0.1 x SSC, 0.1% SDS. Hybridized probes were then detected as previously described [27]. 2.8. Statistical procedure Comparisons between more than two groups were performed by one-way analysis of variance (ANOVA). Bonferroni P values (i.e. corrected for multiple comparisons) are reported. 3. Results Phosphatidylcholine (PC) biosynthesis was measured in blood platelets obtained from 13 CF patients, both with and without pancreatic insufficiency (Table 1). Using U-[‘4C]choline as the substrate, the rate of incorporation into PC was 286 f 74 pmoyh per mg protein. There were no significant differences observed in the rates of incorporation of choline between blood platelets from those CF patients with pancreatic insufficiency and from those CF patients with pancreatic sufficiency. Blood platelets from an age-matched group of healthy patients, in contrast to those of the CF patients’ platelets, exhibited a rate of [ 14C]choline incorporation into PC of only 110 f 33 pmol/h per mg protein. This was less than half the rate of incorporation in the CF platelets, with a significance of P < 0.001 by one-way ANOVA.

Table 1 A comparison of rates of U-[ “C]choline incorporation into phosphatidylcholine Subject

[ “C]Phosphatidylcholine (pmol/h per mg protein)

Normal controls (n = 17) Cystic fibrosis patients (n = 13) Obligate heteroaygotes for cystic fibrosis (n = 5) Patient controls (n = 3)

110 f 33a 286 EIZ74b*d 160 f 41C 103 * 14c

in blood platelets

%lues are given as the means f SD. By one-way ANOVA, CF values were significantly different from normal controls, patient controls and obligate heterozygotes (Bonferroni P values < 0.01, corrected for multiple comparisons). Controls were not significantly different from each other or from obligate heterozygotes. bSignificantly greater than controls, P < 0.001. CNot significantly different from controls. d Significantly greater than obligate heterozygotes P < 0.01.

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The incorporation of [ “C]choline into PC in all experiments was linear for a minimum of 2 h. Two other groups of individuals were also examined for PC biosynthesis in blood platelets. The first group consisted of patients with chronic respiratory disease, other than CF. The rate of choline incorporation into PC was 103 f 14 pmol/h per mg protein and did not differ from the control patients’ rates. The second group tested consisted of obligate heterozygotes for CF with no known symptoms of the disease. The blood platelets from this group exhibited rates of choline incorporation of 160 f 41 pmol/h per mg protein, a rate approximately intermediate between the control and the CF groups but not significantly different from controls. In addition to blood platelets, cultured skin libroblasts from healthy individuals and from patients with CF were examined for rates of PC biosynthesis. Ten cell lines, five from control and five from CF patients were grown until just contact inhibited. U-[ 14C]choline was added with fresh growth medium and the rate of incorporation into PC was measured. Five cell lines from CF patients exhibited approximately 50% greater rate of incorporation of choline into PC than did the five control cell lines, 2758 f 358 vs. 1629 i 471 pmol/h per mg protein, respectively, (P < 0.05). The CF or normal genotype was confirmed by PCR and Southern blotting on genomic DNA extracted from the above five CF and two normal cell lines (data not shown). DNA from all CF cell lines hybridized with AFSos probe. Three samples also showed hybridization with the wild type probe and appeared as a doublet in ethidium bromide stained agarose gel (data not shown), indicating that these patients were compound heterozygotes of AFSos with one of the more rare mutations. DNA amplified from the two normal cell lines hybridized with the wild type probe only. PC biosynthesis is stimulated by, among other metabolites, CAMP and prostaglandin E2 (PGE3. To determine whether the observed increase in choline incorporation into PC could be attributed to an increased sensitivity to these metabolites, the following experiment was performed. Blood platelets from healthy individuals and CF patients were incubated in the presence and absence of either CAMP or PGEz and the rates of U-[ “C]choline incorporation were measured. The ratio of the rates of choline incorporation by cells stimulated by either CAMP or PGEz to those not stimulated are presented in Table 2. These ratios were virtually identical in platelets

Table 2 Stimulation of U-[ 14C]choline incorporation into phosphatidylcholine in blood platelets by CAMP and prostaglandin E2 Subjects

Normal controls (n = 3) CF patients (n = 5)

[‘4C)Phosphatidylcholine (pmol/h per mg protein)

Stimulation ratios

Control

+cAMP

+PGE*

+cAMP/ control

+PGE,I control

251 * 37 630 f 91

510 f 52 1272 zt 140

589 zt 73 1531 f 140

I .98 2.02

2.29 2.43

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Table 3 Phospholipid composition of platelet membranes Subjects

Normal Controls (n = 3) CP Patients (n = 3)

% Total phospholipida PI

PC

PE

PG

PS

LPGb

3.2 3.5

40.2 39.8

30.5 32.1

3.4 3.2

8.0 8.5

17.5 18.0

*Values are the percent of each phospholipid to total measured phospholipids and are means of three experiments; S.E. < f 10% of the means in all cases. bPI, Phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidyl-ethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; LPC, lysophosphatidybholine.

from both CF patients and normal individuals. Thus, the observed increase in choline incorporation into PC in CF patients does not appear to be due to an increased sensitivity to either CAMP or PGE2. The observed increased incorporation of choline has the potential of altering the ratio of the various phospholipid species in the membranes of blood platelets from CF patients. To determine whether this was occurring, the phospholipid compositions of blood platelets from several healthy individuals and several CF patients were measured and compared. Table 3 shows that the proportions of the major phospholipids, PC, PI, PE, PG, PS and LPC were identical in the control and CF individuals tested. Furthermore, the total amounts of phospholipid in platelets of control and CF patients were identical. The failure to detect any increased accumulation or change in the proportion of PC to other phospholiplds in platelets of CF patients suggests that the increased rate of choline incorporation into this phospholipid reflects an increase in its turnover rate and not net synthesis. 4. Discussion

Despite the great progress in understanding the molecular basis of CF, the biochemical connections between the defect in the CFTR Cl- ion channel and the numerous functional abnormalities observed in CF cells remains poorly understood [28]. For example, the relationship between the Cl- ion channel defect, the increased Na+ ion reabsorption as observed in respiratory epithelia and the apparently increased activity of an enzyme which adds fatty acids to glycoproteins remains to be clarified. Among the myriad biochemical alterations observed in CF, many reports indicate that CF cells may have abnormalities in membrane phospholipid and fatty acid turnover. The recent report that CFTR participates in the regulation of membrane recycling in pancreatic cell lines [13] indirectly supports this concept. In this study we show that the rate of phosphatidyl choline (PC) turnover is significantly higher in platelets and tibroblasts of CF patients compared with normal or heterozygote subjects. There is an increased rate of incorporation of choline into PC, although no net accumulation of PC and no change in the overall cellular content

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of phospholipids could be demonstrated, thus indicating an increased turnover rate of this tertiary amine in PC. The increased rate of turnover of choline was consistently observed in the patient samples examined and was independent of age, sex or the presence or absence of pancreatic insufficiency in these individuals. Other patients with chronic respiratory disease did not exhibit this increased rate of turnover of PC. Furthermore, when skin fibroblasts obtained by biopsy of CF patients were examined, they also exhibited an increased rate of choline incorporation in PC, although the differences were not as great as those observed in the blood platelets. This observation supports the hypothesis that the CFTR gene may be indirectly involved in the regulation of membrane recycling with variable functional importance in different cells. If this is the case, the apparently unconnected biochemical alterations observed in CF cells may be explained as consequences of a generalized defect in CFTR regulated membrane turnover. References [I] Rommens JM, Iannuzzi MC, Kerem B et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989;245:1059-1065. [2] Riordan JR, Rommens JM, Kerem B et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066-1073. [3] Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Tsui L. Identification of the cystic fibrosis gene: genetic analysis. Science 1989;245:1073-1080. [4] Anderson MP, Rich DP, Gregory RJ, Smith AE, Welsh MJ. Generation of CAMP-activated chloride currents by expression of CFTR. Science 1991;251:679-682. [5] Anderson MP, Gregory RJ, Thompson S et al. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 1991;253:202-205. [6] Di Sant’Agnese PA, Davis PB. Research in cystic fibrosis. N Engl J Med 1976;295:481-485. (71 Bijman J, Quinton PM. Influence of abnormal Cl- impermeability on sweating in cystic fibrosis. Am J Physiol 1984;247(3:3-9. [8] Yankaskas JR, Knowles MR, Gatzy JT, Boucher RC. Persistence of abnormal chloride Ion perrneability in cystic fibrosis nasal epithelial cells in heterologous culture. Lancet 1985;1:954-956. (91 Boucher RC, Stutts MJ, Knowles MR, Cantley L, Gatzy JT. Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 1986;78:1245-1252. [IO] Willumsen NJ, Boucher RC. Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium. Am J Physiol 1991;261:C332-C341. [I I] Lukacs GL, Chang X, Kartner N, Rotstein OD, Riordan JR, Grinstein S. The cystic fibrosis transmembrane regulator is present and functional in endosomes: role as a determinant of endosomal pH. J Biol Chem 1992;267:14568-14572. [I21 Barasch J, Kiss B, Prince A, Saiman L, Gruenert D, Al-Awqati Q. Defective acidification of intracellular organelles in cystic fibrosis. Nature 1991;352:70-73. [I31 Bradbury NA, Jilling T, Berta G, Sorscher EJ, Bridges RJ, Kirk KL. Regulation of plasma membrane recycling by CFTR. Science 1992;256:530-532. [ 141 Hubbard VS, Dunn GD. Fatty acid composition of erythrocyte phospholipids from patients with cystic fibrosis. Clin Chim Acta 1980;102:1IS- I 18. [IS] Rogiers V, Dab I, Michotte Y, Vercruysse A, Crokaert R, Vis HL. Abnormal fatty acid turnover in the phospholipids of the red blood cell membranes of cystic fibrosis patients (in vitro study). Pediatr Res 1984;18:704-709. [I61 Kuo PT, Huang NN, Bassett DR. The fatty acid composition of the serum chylomicrons and adipose tissue of children with cystic fibrosis of the pancreas. J Pediatr 1962;60:394-403.

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