Biomedical subjects

Nam Soo Joo

Publications and source records attributed to Nam Soo Joo.

8 recordsLinked to original sources

PubMed2006

Hyposecretion, not hyperabsorption, is the basic defect of cystic fibrosis airway glands.

Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na(+) channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands.

Amiloride↗

PubMed2004

An inwardly rectifying potassium channel in apical membrane of Calu-3 cells.

Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 +/- 0.5 pS (n = 11), between 0 and -200 mV, and was 9.6 +/- 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of approximately 2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl- secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16-28 mm if 11-25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139-146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+ -K+ -ATPase in Calu-3 cells.

Adenosine Triphosphate↗

PubMed2004

Acid and base secretion in the Calu-3 model of human serous cells.

Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.

Acetazolamide↗

PubMed2004

Regulation of antiprotease and antimicrobial protein secretion by airway submucosal gland serous cells.

Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.

Amino Acid Sequence↗

PubMed2004

A "virtual gland" method for quantifying epithelial fluid secretion.

We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (Jv), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-microl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37 degrees C. Basal Jv was 2.7 +/- 0.1 microl x cm(-2) x h(-1) (n = 42), and TEP was -9.2 +/- 0.6 mV (n = 33); both measures were reduced to zero by ouabain (n = 6) x Jv and TEP were stimulated 64 and 59%, respectively, by 5 microM forskolin (n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone (n = 5), 213 and 122% by 333 nM thapsigargin (n = 5), and 520 and 240% by forskolin + thapsigargin (n = 6). Basal Jv and TEP were inhibited to 82 and 63%, respectively, with 10 microM bumetanide (n = 5), 71 and 82% with 100 microM acetazolamide (n = 5), and 47 and 56% with 600 microM glibenclamide (n = 4). Basal Jv and TEP were 52 and 89% of control values, respectively, after HCO3- replacement with HEPES (n = 16). The net HCO3- concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (approximately 80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.

Cell Line↗

PubMed2004

Submucosal glands and airway defense.

Most airway mucus is produced by submucosal glands in response to neural signals. Gland mucus traps microbes, inhibits their replication, and clears them from the airways. In cystic fibrosis mucus clearance is compromised, allowing pathogens to persist in static mucus. These trigger an influx of inflammatory cells, but optimal effectiveness of inflammation, and especially its resolution, also requires effective mucus clearance. Our objective is to understand the basis for defective mucus clearance in cystic fibrosis. We discovered that in subjects with cystic fibrosis, submucosal gland secretion in response to agents that elevate intracellular cyclic AMP level is completely lost and mucus stimulated by elevating intracellular Ca2+ level is thicker. We hypothesize that loss of functional cystic fibrosis transmembrane conductance regulator from gland serous cells renders them unable to secrete anions and fluid in response to any stimulus, resulting in thickened gland mucus that can be tethered to the gland ducts. In primary ciliary dyskinesias, mucus is normal, but the dysfunctional cilia lining the gland ducts may also lead to inadequate clearance of mucus from glands. Thus, understanding of lung pathology in each disease may require that an improved understanding of gland structure and function be added to our rapidly growing understanding of surface epithelia.

Animals↗

PubMed2002

Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands.

We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP](i) (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n = 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP](i) suggests that altered secretion of gland mucus could contribute to CF lung disease.

Adult↗

PubMed2002

Mucus secretion from single submucosal glands of pig. Stimulation by carbachol and vasoactive intestinal peptide.

Secretion rates of >700 individual glands in isolated tracheal mucosa from 56 adult pigs were monitored optically. "Basal" secretion of 0.7 +/- 0.1 nl x min(-1) gland(-1) was observed 1-9 h post-harvest but was near zero on day 2. Secretion to carbachol (10 microm) peaked at 2-3 min and then declined to a sustained phase. Peak secretion was 12.4 +/- 1.1 nl x min(-1) gland(-1); sustained secretion was approximately one-third of peak secretion. Thapsigargin (1 microm) increased secretion from 0.1 +/- 0.05 to 0.7 +/- 0.2 nl x min(-1) gland(-1); thapsigargin did not cause contraction of the trachealis muscles. Isoproterenol and phenylephrine (10 microm each) were ineffective, but vasoactive intestinal peptide (1 microm) and forskolin (10 microm) each produced sustained secretion of 1.0 +/- 0.5 and 1.7 +/- 0.2 nl x min(-1) gland(-1), respectively. The density of actively secreting glands was 1.3/mm(2). Secretion to either carbachol or forskolin was inhibited (approximately 50%) by either bumetanide or HCO(3)(-) removal and inhibited approximately 90% by the combined treatments. Mucus secreted in response to carbachol or forskolin was acidic by approximately 0.2 pH units relative to the bath and remained acidic by approximately 0.1 pH units after bumetanide. The strong secretory response to vasoactive intestinal peptide, the acidity of [cAMP](i)-stimulated mucus, and its inhibition by bumetanide were unexpected.

Animals↗