In porcine bronchi, inhibition of both Cl- and HCO3- transport is required to block the anion secretion response to ACh and to cause mucus accumulation within ACh-treated submucosal gland ducts [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997]. In this previous study, a combination of three potential HCO3- transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl-/HCO3- exchange, and Na+/H+ exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced HCO3- secretion in airway glands by determining which of the three inhibitors, in combination with bumetanide, is required to block anion secretion and so cause ductal mucin accumulation. Gland duct mucin content was measured in distal bronchi isolated from domestic pigs. Addition of either bumetanide alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no significant effect on ACh-induced mean gland duct mucin content. In contrast, glands treated with bumetanide plus DMA as well as glands treated with all four anion transport blockers were almost completely occluded with mucin after the addition of ACh. These data suggest that mucin is cleared from the ducts of bronchial submucosal glands by liquid generated from Cl(-)- and DMA-sensitive HCO3- transport.
"The pig bronchial epithelium is pseudostratified and contains ciliated, basal, and goblet cells, and abundant submucosal glands (reviewed in ref. 12). Importantly, the distribution of submucosal glands in the conducting airways and the CFTR-dependent and -independent secretion of liquid and macromolecules is similar to human submucosal glands.13,14,15,16 Thus, pig airway cell composition is much more akin to human airways than are those of mice. "
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012.
"Because normal submucosal glands are capable of secreting HCO 3 − (Inglis et al., 1998; Trout et al., 1998a) and the CFTR is conductive to this anion (Poulsen et al., 1994), there has been speculation that CFTR might be required for normal HCO 3 − secretion by airway glands. Jayaraman et al. (2001) used fluorescent pH-sensitive probes injected directly into liquid droplets as they emerged from the gland ducts to measure gland fluid pH. "
[Show abstract][Hide abstract] ABSTRACT: Submucosal glands of the tracheobronchial airways provide the important functions of secreting mucins, antimicrobial substances, and fluid. This review focuses on the ionic mechanism and regulation of gland fluid secretion and examines the possible role of gland dysfunction in the lethal disease cystic fibrosis (CF). The fluid component of gland secretion is driven by the active transepithelial secretion of both Cl(-) and HCO(3)(-) by serous cells. Gland fluid secretion is neurally regulated with acetylcholine, substance P, and vasoactive intestinal peptide (VIP) playing prominent roles. The cystic fibrosis transmembrane conductance regulator (CFTR) is present in the apical membrane of gland serous cells and mediates the VIP-induced component of liquid secretion whereas the muscarinic component of liquid secretion appears to be at least partially CFTR-independent. Loss of CFTR function, which occurs in CF disease, reduces the capacity of glands to secrete fluid but not mucins. The possible links between the loss of fluid secretion capability and the complex airway pathology of CF are discussed.
"ed basolateral Cl − conductance has not been described in primary exocrine cells , and the pre - sence of one here would make this cell unique among exocrine fluid - secreting cells . Thus , the simplest and most logical explanation for our data , as well as the explanation that agrees with observations of intact mouse , porcine and human glands ( Inglis et al . 1997b , 1998 ; Trout et al . 1998 , 2001 ; Ballard et al . 1999 ; Jayaraman et al . 2001 ; Joo et al . 2001a , b , 2002b , 2006 ; Song & Verkman , 2001 ; Thiagarajah et al . 2004 ; Salinas et al . 2005 ; Song et al . 2005 ; Wu et al . 2006 ; Ianowski et al . 2007 ) , is that the CCh - stimulated Cl − efflux pathway we have observed is an apical memb"
[Show abstract][Hide abstract] ABSTRACT: Airway submucosal glands are sites of high expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and contribute to fluid homeostasis in the lung. However, the molecular mechanisms of gland ion and fluid transport are poorly defined. Here, submucosal gland serous acinar cells were isolated from murine airway, identified by immunofluorescence and gene expression profiling, and used in physiological studies. Stimulation of isolated acinar cells with carbachol (CCh), histamine or ATP was associated with marked decreases in cell volume (20 +/- 2% within 62 +/- 5 s) that were tightly correlated with increases in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) as revealed by simultaneous DIC and fluorescent indicator dye microscopy. Simultaneous imaging of cell volume and the Cl(-)-sensitive fluorophore SPQ indicated that the 20% shrinkage was associated with a fall of [Cl(-)](i) from 65 mm to 28 mm, reflecting loss of 67% of cell Cl(-) content, accompanied by parallel efflux of K(+). Upon agonist removal, [Ca(2+)](i) relaxed and the cells swelled back to resting volume via a bumetanide-sensitive Cl(-) influx pathway, likely to be NKCC1. Accordingly, agonist-induced serous acinar cell shrinkage and swelling are caused by activation of solute efflux and influx pathways, respectively, and cell volume reflects the secretory state of these cells. In contrast, elevation of cAMP failed to elicit detectible volume responses, or enhance those induced by submaximal [CCh], because the magnitude of the changes were likely to be below the threshold of detection using optical imaging. Finally, when stimulated with cholinergic or cAMP agonists, cells from mice that lacked CFTR, as well as wild-type cells treated with a CFTR inhibitor, exhibited identical rates and magnitudes of shrinkage and Cl(-) efflux compared with control cells. These results provide insights into the molecular mechanisms of salt and water secretion by lung submucosal glands, and they suggest that while murine submucosal gland fluid secretion in response to cholinergic stimulation can originate from CFTR-expressing serous acinar cells, it is not dependent upon CFTR function.
The Journal of Physiology 09/2007; 582(Pt 3):1099-124. DOI:10.1113/jphysiol.2007.131995 · 5.04 Impact Factor
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