Erythrocyte membrane ATP binding cassette (ABC) proteins: MRP1 and CFTR as well as CD39 (ecto-apyrase) involved in RBC ATP transport and elevated blood plasma ATP of cystic fibrosis.

In addition to the better-known roles of the erythrocyte in the transport of oxygen and carbon dioxide, the concept that the red blood cell is involved in the transport and release of ATP has been evolving (J. Luthje, Blut 59, 367, 1989; G. R. Bergfeld and T. Forrester, Cardiovasc. Res. 26, 40, 1992; M. L. Ellsworth et al., Am. J. Physiol. 269, H2155, 1995; R. S. Sprague et al., Am. J. Physiol. 275, H1726, 1998). Membrane proteins involved in the release of ATP from erythrocytes now appear to include members of the ATP binding cassette (ABC) family (C. F. Higgins, Annu. Rev. Cell Biol. 8, 67, 1992; C. F. Higgins, Cell 82, 693, 1995). In addition to defining physiologically the presence of ABC proteins in RBCs, accumulating gel electrophoretic evidence suggests that the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance-associated protein (MRP1), respectively, constitute significant proteins in the red blood cell membrane. As such, this finding makes the mature erythrocyte compartment a major mammalian repository of these important ABC proteins. Because of its relative structural simplicity and ready accessibility, the erythrocyte offers an ideal system to explore details of the physiological functions of ABC proteins. Moreover, the presence of different ABC proteins in a single membrane implies that interaction among these proteins and with other membrane proteins may be the norm and not the exception in terms of modulation of their functions.

Immortalized intrahepatic mouse biliary epithelial cells: immunologic characterization and immunogenicity.

Nonsuppurative destructive cholangitis (NSDC), a process of T-cell-mediated destruction of biliary epithelia observed in primary biliary cirrhosis (PBC), graft-versus-host disease (GVHD), and hepatic allograft rejection (HAR), also occurs in the B10. D2-->BALB/c model of GVHD. To advance studies of immunopathogenesis in this murine model, we immortalized 4 BALB/c intrahepatic biliary epithelial cell (BEC) lines as a reliable source of target cells. Freshly isolated BEC, as well as each cell line, expressed cytokeratin-19 (CK-19), epithelial cell adhesion molecule (EPCAM) and cystic fibrosis transmembrane conductance regulator (CFTR). None expressed albumin. Immortalized cells also expressed SV40 large T antigen. Class I major histocompatibility complex (MHC) was expressed by >97% of immortalized cells, while class II MHC and intercellular adhesion molecule-1 (ICAM-1) expression ranged from 0% to 13% and 14% to 74%, respectively. Interferon gamma (IFN-gamma) induced aberrant class II MHC expression and increased expression of ICAM-1. Variable proportions of immortalized cells expressed B7-1/B7-2 molecules and FAS. IFN-gamma significantly reduced B7-1 expression in some lines and significantly increased B7-2 expression in others. Allografts of freshly isolated and immortalized BEC injected into subscapular fat pads spontaneously formed duct-like structures. Inflammation was absent in BALB/c recipients. In contrast, inflammatory lesions in B10.D2 recipients were reminiscent of NSDC. Our results indicate that BALB/c-immortalized intrahepatic biliary cells: 1) retain the phenotype of mouse BEC; 2) can be induced to express aberrant class II MHC and increased ICAM-1; 3) express costimulatory B7-1/B7-2 molecules and FAS; and 4) spontaneously form duct-like structures after in vivo injection that are immunogenic in B10.D2 mice. These cell lines should facilitate future studies of the immunopathogenesis of NSDC in the B10. D2-->BALB/c murine model.

Development of salivary gland cell lines for studies of signaling and physiology.

We developed and characterized an immortalized rat parotid cell line to use in salivary gland studies. The cells were immortalized by retroviral transduction of SV40 large T antigen into isolated parotid cells. Using immunocytochemical techniques, we found that the immortalized epithelial cells were ductal, rather than acinar, in nature. Cells were grown under coculture conditions with lethally irradiated NIH3T3 cells. One cell line, which was designated RPG1/SV40 cells (for rat parotid gland 1/SV40 transformant), was selected for characterization. These cells formed a sheet epithelium with tight junctions and a measurable transepithelial resistance. RPG1/SV40 cells responded to muscarinic receptor (carbachol) and/or P2 purinoceptor (ATP and UTP) stimuli with increases in the following: (1) intracellular free-calcium concentration ([Ca2+]i); (2) the short-circuit current (ISC) across the epithelium; (3) the tyrosine phosphorylation of PKC delta; and (4) MAP kinase activity. Thus, the cells appear to be useful for a wide range of studies involving physiology, biochemistry, and signal transduction approaches.

Dysregulation of proteoglycan production by intrahepatic biliary epithelial cells bearing defective (delta-f508) cystic fibrosis transmembrane conductance regulator.

Hepatic dysfunction in cystic fibrosis (CF) has been attributed to accumulation of viscous mucoid secretions in intrahepatic bile ducts. The purpose of our study was to compare glycoconjugate secretion by intrahepatic biliary epithelial (IBE) cells derived from normal livers and livers of CF patients with the delta F508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Confluent cells were incubated with 3H-glucosamine (GlcN) for 16 hours, and radiolabeled macromolecules were analyzed for the amount and type of glycoconjugates. Incorporation of 3H-GlcN into macromolecular glycoconjugates was two- to threefold higher in CF cells versus normals, as was uptake of 3H-Glcn into the cytoplasm of CF cells. Gel exclusion chromatography on Sepharose Cl 4B revealed that the secreted glycoconjugates from CF cells eluted entirely in the excluded fraction (molecular weight > 2 x 10(6)), while, in the normal cells, 60% of the glycoconjugates eluted as lower-molecular-weight species. The high-molecular-weight glycoconjugates in both CF and normal cells were identified as chondroitin sulfates, as evidenced by susceptibility to beta elimination, chondroitinase digestion, and amino acid composition. Western blotting of IBE cell secretions with a polyclonal antibody to chondroitin sulfate revealed proteoglycan bands at 100 and 210 kd. Our results indicate that secretion of chondroitin sulfate is markedly increased in CF biliary epithelium in vitro compared with non-CF cells. Increased uptake of precursor 3H-GlcN may contribute to enhanced glycosylation of chondroitin sulfate in CF cells.

Autosomal dominant polycystic kidney disease decreases anion exchanger activity.

Liver cysts, the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD), derive from the intrahepatic biliary epithelium (IBE) and are found in 60-75% of ADPKD patients on dialysis. Secretin-induced secretion by the normal IBE is rich in HCO3-, whereas intact ADPKD liver cysts secrete primarily Cl- in response to secretin. To evaluate the mechanisms of decreased HCO3- secretion by ADPKD liver cysts, we utilized SV40 large T antigen-immortalized normal IBE and ADPKD liver cyst-derived epithelia (LCDE) cell lines that we created. These cell lines express biliary but not hepatocyte markers. Anion exchanger (AE) function was assessed by the response of intracellular pH (pHi) to acute Cl- removal. 2',7'-Bis(carboxyethyl)-5-(6)-carboxyfluorescein-loaded monolayers were continuously perfused with physiological HCO3- buffer containing Cl- or gluconate. In IBE cell line H75 (n = 6), acute Cl- removal alkalinized pHi at a rate of 0.04 +/- 0.01 min-1. AE function was significantly decreased in LCDE cell line CL3 (n = 6) to a rate of 0.01 +/- 0.01 min-1 after Cl- removal. Northern blot analysis demonstrated equivalent levels of AE2 mRNA in both cell lines. AE1 mRNA was undetectable. Immunoblot analysis demonstrated the AE2 polypeptide in both cell lines, but the level of mature glycosylated AE2 polypeptide was reduced in LCDE cells. Immunofluorescence microscopy demonstrated decreased membrane-localized AE2 in LCDE cells. These findings suggest that decreased plasmalemmal AE2 may account for decreased AE function in LCDE cells and suggest a possible explanation for decreased secretion of HCO3- by ADPKD liver cysts.

An in vitro model of infection of human biliary epithelial cells by Cryptosporidium parvum.

Cryptosporidium parvum infection in the immunosuppressed host is frequently complicated by biliary tract involvement. The recent production of human biliary epithelial cell lines was exploited to develop an in vitro model of biliary cryptosporidiosis. Infection with C. parvum oocysts was detected by IFA and ELISA and confirmed by transmission electron microscopy. Inoculation of monolayers with 10(4) to 5 X 10(5) oocysts/well resulted in a dose-dependent increase in infection. Time-course experiments showed that the number of parasitic stages was maximal at 18-24 h after inoculation. Infection was significantly enhanced by bile at concentrations of 50 and 100 microg/mL and inhibited by 400 microg/mL paromomycin. Infection of human biliary cells with C. parvum can be consistently achieved and monitored by use of IFA or ELISA. This system will be of use in evaluating mechanisms of C. parvum infection and response to therapeutic agents in biliary cryptosporidiosis.

A descriptive survey of pediatric human immunodeficiency virus-infected long-term survivors.

OBJECTIVE: To identify the population of human immunodeficiency virus-infected pediatric long- term survivors (LTS) followed in major medical institutions in California, Florida and New Jersey. METHODS: A cross-sectional survey was performed with data collection forms sent to all investigators. Demographic, clinical, and laboratory data were obtained on all living patients >/=8 years infected in the perinatal period with human immunodeficiency virus. RESULTS: A total of 143 perinatally infected and 54 children infected by neonatal transfusion were identified. Fifty-four children (27%) had absolute CD4 counts >/=500 cells/mm (group 1: mean age 9.8 years), 54 children (27%) had CD4 counts between 200 and 500 cells/mm (group 2: mean age 10.1 years), and 89 children (45%) had CD4 counts <200 cells/mm (group 3: mean age 10.4 years). Ninety-five (48%) patients had developed AIDS defining conditions; 14 (26%) in group 1, 26 (48%) in group 2, and 55 (62%) in group 3. Ninety-two percent of patients had received antiretrovirals. Perinatally human immunodeficiency virus-infected children tended to be younger (mean age 9.8 years) than children infected via a blood transfusion (mean age 11 years). Generalized lymphadenopathy was the most prevalent clinical finding. Lymphoid interstitial pneumonia and recurrent bacterial infections were the most prevalent acquired immune deficiency syndrome-defining conditions. Twenty percent of LTS had CD4 counts >/=500 cells/mm and no immune deficiency syndrome-defining conditions. CONCLUSIONS: Pediatric LTS were in variable stages of disease progression. The proportion of children within each CD4 strata did not differ by mode of acquisition of infection. Increased CD4 counts were inversely proportional to age. Only 20% of pediatric LTS had minimal to no disease progression.

Cystic fibrosis hetero- and homozygosity is associated with inhibition of breast cancer growth.

Cystic fibrosis (CF) is the most common lethal recessive genetic disease of the Caucasian population. Although reports of cancer frequency in CF have emphasized an elevated observed-to-expected ratio of 6.5 for digestive tract cancers, these studies also show a significantly decreased observed-to-expected ratio for other malignancies including breast cancer. The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATP channel. We found that heterozygous and homozygous CFTR knockout mice had elevated blood ATP concentrations. Elevated extracellular ATP is known to inhibit tumor growth in vivo and in vitro. Using double mutant mice created by F2 generation crosses of CFTR knockout and nude mice, we observed reduced breast tumor implantability in CFTR homozygous nude animals. Decreased tumor growth rate was observed in both CFTR heterozygous and homozygous nude animals. Extracellular ATP reduced human breast tumor cell growth rate in vitro, and a breast tumor transfected with human CFTR that had high extracellular ATP concentrations in vitro correspondingly had a slower growth rate in vivo. The results suggest that both CFTR heterozygosity and homozygosity suppress breast cancer growth and that elevated extracellular ATP can account for this phenomenon.

Pediatric treatment update.

AIDS: The ACTG 152 study compared AZT monotherapy with ddI monotherapy in HIV-positive children, ranging in age from 3 months to 18 years. The Data Safety and Monitoring Board (DSMB) recommended that AZT monotherapy be discontinued based on survival and HIV disease progression. ACTG 152 opened in August 1991 and included 839 children. The trial assessed the efficacy of three antiretroviral drugs as first-line treatment. Only asymptomatic children with relatively normal laboratory values were excluded, so the trial was unusually broad. There were fewer toxicities associated with ddI monotherapy than ddI plus AZT. Final results clearly support the results of ACTG 175, which found that AZT alone is not the most effective first-line therapy. Pneumocystis carinii pneumonia (PCP) is the most common opportunistic infection occurring in HIV-positive children. However, CD4 cell counts are not an effective indicator of PCP risk until the children are twelve months old. In March 1994, the National Pediatric and Family HIV Resource Center, in conjunction with the Centers for Disease Control and Prevention (CDC), revised the guidelines for pediatric care. The new guidelines recommend that all HIV-positive infants, or those born to HIV-positive mothers, be started on prophylactic treatment at the age of one month.^ieng

Immortalized human bladder cell line exhibits amiloride-sensitive sodium absorption.

We have produced a continuous cell line using retroviral transduction of simian virus 40 (SV40) large T antigen into epithelial cells grown from a cystoscopic bladder biopsy from a female patient with interstitial cystitis. Immortalized urothelial cells are grown in a hormonally supplemented medium in the presence of lethally irradiated NIH-3T3 fibroblast coculture. They maintain their epithelial appearance and are positive for cytokeratins. SV40 large T antigen is localized to the cell nucleus. When grown on Anocell permeable supports, the cells form a complex epithelium with scalloped luminal membranes, apical junctional complexes containing tight junctions, stratification, transepithelial resistance of 500-1,000 omega.cm2, amiloride-sensitive short-circuit current indicative of active transepithelial Na+ absorption, and functional evidence for basolateral Na-K-adenosinetriphosphatase. This immortalized bladder cell line will facilitate the study of human bladder epithelial function and the response to diverse physiological and pathophysiological stimuli.

Matrix metalloproteinase activity in human intrahepatic biliary epithelial cell lines from patients with autosomal dominant polycystic kidney disease.

Hepatic cysts derived from intrahepatic bile ducts are the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD). Cyst enlargement involves cell proliferation, fluid secretion into cysts, and alterations in extracellular matrix. To study hepatic cyst formation, continuous cell lines from human normal intrahepatic biliary epithelium (IBE) and ADPKD liver cyst-derived epithelium (LCDE) were developed. Because matrix degradation and remodeling are important for cyst formation and growth, we investigated matrix modifying enzymes expressed in IBE and LCDE cell lines. Gelatin substrate zymography showed that two matrix degrading activities with characteristics of matrix metalloproteinases are secreted from these cell lines. Western immunoblotting suggests that these activities correspond to the 72 kDa (Gelatinase A) and 92 kDa (Gelatinase B) type IV collagenases. Although the level of Gelatinase A activity is comparable in both IBE and LCDE cell lines, Gelatinase B activity is substantially increased in LCDE lines.

Continuous epithelial cell lines from ADPKD liver cysts exhibit characteristics of intrahepatic biliary epithelium.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into epithelial cells removed from the lumen of liver cysts from four female patients with autosomal dominant polycystic kidney disease (ADPKD). Liver cyst-derived epithelial (LCDE) cell lines are grown in a hormonally supplemented medium in the presence of lethally irradiated NIH/3T3 fibroblast coculture. LCDE cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratin 7 and 19 and gamma-glutamyl transpeptidase while being negative for hepatocyte markers. SV40 large T antigen is localized to the cell nucleus. LCDE cells have been grown continuously for periods exceeding 12 mo and 25 passages (170 population doublings). LCDE cells exhibit intracellular pH regulatory pathways that, with one exception, are similar to those found in normal intrahepatic biliary epithelium. These LCDE cell lines exhibit impaired alkalinization in response to Cl- substitution. This finding is suggestive of decreased function or abundance of a Cl-/HCO3- anion exchanger and could account for the failure of ADPKD hepatic cysts to secrete HCO3- in response to secretin.

Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines.

The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P2U-purinoceptor stimulation which is consistent with the opening of a Ca(2+)-sensitive Cl- channel. The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.

Older children and adolescents living with perinatally acquired human immunodeficiency virus infection.

OBJECTIVE: To describe the clinical, immunologic, and psychosocial characteristics of children living with perinatally-acquired human immunodeficiency virus (HIV) infection beyond the age of 9 years. METHODS: This is a descriptive cohort study of 42 surviving perinatally infected children older than 9 years followed at the Children's Hospital Acquired Immunodeficiency Syndrome (AIDS) Program (part of a university-based inner city medical center) as of June 1993. The study is based on medical record data of clinical, immunologic, and psychosocial parameters. RESULTS: The cohort includes 20 boys and 22 girls with a mean age of 136 months. The mean age at diagnosis of HIV infection was 88 months, and 59.5% were asymptomatic at the time of diagnosis. Currently, after a mean follow-up period of 48 months from diagnosis, 23.8% remain asymptomatic, 19.1% have non-AIDS-defining HIV-related symptoms, and 57.1% have AIDS; 85.7% of the cohort did not develop HIV-related symptoms until after 48 months of age (late-onset prolonged survivors). There was an average annual decline of 71.4 CD4+ cells/microL in the cohort from the ages of 7 to 16 years, and 21.4% have a current CD4+ lymphocyte count of greater than 500 cells/microL, 28.6% between 200 and 500 cells/microL, and 50% less than 200 cells/microL; 76% are orphaned as a result of maternal death, with the majority of the cohort (60%) cared for by extended family members. Disclosure of diagnosis has occurred in 57.1%. The vast majority of the cohort (76%) are attending regular school, with the remainder in special education. CONCLUSIONS: Although close to one quarter of the children and adolescents ages 9 to 16 years living with perinatally acquired HIV infection described in this cohort remain asymptomatic and have a relatively intact immune system, the remainder are living with significant HIV-related symptoms, many of which are chronic in nature and have an impact on daily living. The children in this cohort had both significant immunologic deterioration and symptomatic disease progression during the mean follow-up period of 48 months from the time of diagnosis with HIV infection.

Correction of the cystic fibrosis defect by gene complementation in human intrahepatic biliary epithelial cell lines.

BACKGROUND/AIMS: Hepatobiliary disease is the second most common cause of mortality in patients with cystic fibrosis (CF). In the liver, only the intrahepatic biliary epithelial (IBE) cells express cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine whether human CF-derived IBE cells can be infected with adenovirus and the CF phenotype complemented. METHODS: IBE cells were isolated from 2 patients with CF and immortalized using retrovirus transduction of SV40 large T antigen. Immortalized cells were infected with the adenovirus vector Ad2/CFTR2 and assayed 2-31 days postinfection for cyclic adenosine monophosphate (cAMP)-induced halide efflux. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe 6-methoxy-N-(3-sulfopropyl)-quinolinium. RESULTS: CF-derived IBE cell lines express biliary specific markers and express no cAMP-inducible halide efflux. Following infection with the adenovirus vector Ad2/CFTR2, a cAMP-induced halide efflux was observed for 31 days, although the number of responsive cells decreased with time. CONCLUSIONS: Human CF-IBE cells can be infected by adenovirus and the defective CFTR complemented. The loss of responsive cells with time could be due to loss of construct and/or a reduced growth of cells that are overexpressing CFTR. These CF-IBE cell lines offer an opportunity to determine the mechanisms responsible for hepatobiliary disease in the patients with CF.

Experimental renal failure in the rat modulates cardiac Na,K-ATPase alpha 2 mRNA but not protein.

The decreased abundance and enzymatic activity of myocardial Na,K-ATPase have been recognized previously to occur in chronic uremia. However, the activity of the cardiac sodium pump as defined by the uptake of 86Rb is normal. The discrepancies between these findings may have resulted from the inability to distinguish between the different Na,K-ATPase isoforms now known to exist in cardiac muscle. To investigate this question, steady-state levels of Na,K-ATPase alpha and beta mRNA isoforms, alpha 1, alpha 2, and beta 1 protein, and specific high-affinity binding of [3H]ouabain were quantitated in cardiac muscle from uremic and pair-fed, sham-operated control rats. Steady-state levels of alpha 2 and beta 2 mRNA were significantly decreased (percentage of control levels: alpha 2, 48 +/- 10; beta 2, 74 +/- 9; N = 10; P < 0.025) in chronic renal failure without any change in alpha 1, alpha 3, or beta 1 expression. The number of high-affinity [3H]ouabain-binding sites and Na,K-ATPase alpha 1, alpha 2, and beta 1 subunits was not different from control. In acute renal failure, alpha 2 and beta 2 mRNA levels also were significantly decreased (percentage of control levels: alpha 2, 24 +/- 5; beta 2, 44 +/- 8; N = 6; P < 0.001), but there was no change in the level of alpha 3 or beta 1 mRNA, the number of high-affinity [3H]ouabain-binding sites, or the level of Na,K-ATPase alpha 2 and beta 1 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.

Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines.

Continuous epithelial cell lines from individuals with cystic fibrosis (CF) and normal controls are required to understand the genetic and cellular defects in CF. We used retroviruses to transduce SV40 large T antigen into nasal epithelial cells. Transformed continuous cell lines were isolated that expressed epithelial markers, cytokeratin, and tight junctions. Northern blot analysis shows that all of the cell lines express the putative CF gene mRNA. Studies of transepithelial electrolyte transport show that CF and normal cell lines develop a transepithelial electrical resistance. Normal but not CF cell lines secreted Cl- in response to agonists that increase cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (isoproterenol, forskolin, and a membrane-permeant analogue of cAMP) or in response to a tumor-promoting phorbol ester that activates protein kinase C. In contrast, the Ca2(+)-elevating agonist bradykinin and the Ca2+ ionophore A23187 stimulated secretion in both normal and CF cell lines. The continuous cell lines we have produced maintain their proper phenotypes and will serve as useful tools in understanding the pathophysiology of CF.