Anthony Paradiso
Research Assistant Professor of Medicine

Degrees
     BS: 
      (1979) WA State U., Pullman, WA
     MS: 
       (1981) WA State U., Pullman, WA
     PhD:
         (1985) U. of CA, Berkeley, CA

    

Office:   6007B Thurston-Bowles
Phone:   919-966-9142
Fax:        919-966-7524
email:   paradiso@med.unc.edu

Background and Research Goals of Our Laboratory

Airway surfaces are lined with a layer of secretions composed primarily of water, ions, and macromolecules generated, we speculate, locally by surface epithelial cells and/or submucosal glands. The depth of ASL is very shallow (5 to 100 m m), which is essential for maintaining low resistive airflow, but makes studies of ASL physiology technically challenging. The pH of liquids elaborated on airway epithelial surfaces potentially

influences many processes including, ion channels, mucus adhesion to biological membranes, attachment of bacterial and viral pathogens to mucus, and innate immune system. Surprisingly, the pH of airway surface liquid (ASL) has not been comprehensively studied, and the processes important for regulation ASL pH are definitively not known. Moreover, ASL can be partitioned into three open compartments, a glycocalyx coating the apical cell surface, a pericilary liquid (PCL) layer through which cilia beat freely, and an overlying mucus layer that entraps particles to be transported by coordinated ciliary movement (see Fig.1).

In healthy airways, ASL depth, ionic composition and viscoelastic properties of the mucus gel are optimally regulated for efficient mucociliary clearance. In contrast to the healthy airway, cystic fibrosis (CF) airways are characterized by thick mucus and reduced depth of the PCL compartment, thereby hampering airway clearance mechanisms (cough/mucociliary movement) from removing inhaled toxins from the airway (see Fig.2). Furthermore, the carbohydrate-rich glycocalyx contains intrinsic glycolipids and glycoproteins

as well as adsorbed glycoproteins and proteoglycans. In human airway epithelia, the apical cell surface is abundant in this glycocalyx, and the pH within the glycocalyx is predicted to be low because of electrostatic effects arising from the fixed negative charges of sugar moieties and membrane phospholipid head groups. For example, glycoaminoglycans as well as mucin-type high molecular weight glycoconjugates (HMGs) released spontaneously from epithelial cells are heavily sialiated and sulfated in airway cells. A major consequence of sialiation and sulfation is that the negative surface potential of HMGs results in the accumulation of cations on their surfaces. In a pH –sensitive biomolecular system such as an anionic glycocalyx and mucus gel, the negative potential of these groups will result in a drop in local

perimolecular pH with greater protonation of fixed anionic charges (buffers) associated with HMGs. Indeed, if the density of the negative fixed buffers are different between the cell surface glycocalyx, the PCL layer and the mucus gel, then it is expected that the pH within these compartments will be different and, therefore, pH gradients will be established between these compartments.

Ongoing research in our laboratory is studying the hypothesis that regulation of airway ASL pH is defective in CF proximal human airway epithelia. It is well known that a major consequence of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the pancreas is reduced HCO₃^- and pH of

pancreatic secretions. However, the effect of CFTR dysfunction on ASL pH is not well documented. This information is crucial to explain the pathophysiology of CF in the airway because, as noted above, the binding of pathogens and the physical properties of mucus are expected to be pH sensitive. Our preliminary data (see Fig. 3) indicate that bulk ASL pH is more acidic in CF compared to healthy airway cells, and we speculate that this abnormality is exaggerated in the near apical membrane extracellular compartments of airway epithelial cells.

We hypothesize that in healthy airways, ASL pH is regulated by parallel operation of apically-located H⁺,K⁺-ATPase(s), CFTR-mediated HCO₃^- flux, and HCO₃^- movement via the paracellular shunt (see Fig.4).

We further hypothesize that fixed negative charges within the glycocalyx coating the apical cell surface, the PCL layer, and the overlying mucus gel profoundly influence local pH within microdomains of ASL by acting as sinks for H+. Finally, we hypothesize that the lack of CFTR-mediated HCO3- secretion coupled with a constitutively active H+,K+-ATPase, render ASL compartments abnormally acidic in CF airway epithelia and, consequently, increase the adhesion of the mucus gel to airway epithelial surfaces. One direction of our research is to use pH-sensitive fluorescent markers to identify H+ and HCO3- transport routes that have the capacity to modify ASL pH in airway cells in vitro.

A second goal our research will employ confocal microscopy in conjunction with unique near-membrane fluorescent probes to test whether pH gradients are formed within microdomains (i.e., glycocalyx, PCL and mucus gel) of ASL during volume absorption in vitro (see Fig. 5). For the in vitro studies, we employ

a novel culture system of well-differentiated human nasal and bronchial epithelial cells from normal donors and CF patients. The final goal of our research will employ pH microelectrode techniques and chemical assays to measure nasal pH in vivo in normal and CF subjects. With protocols designed on the basis of our in vitro data, we are testing the hypothesis that surface epithelia abnormally regulate pH in CF subjects in vivo, and we plan to extend this characterization

to include the contribution of pH regulators, especially to the abnormality in CF, to glandular secretion. Our long-term goal is to elucidate the pathogenic steps leading to CF airway disease with loss of CFTR function.

1. Paradiso, A.M., C.M.P. Ribeiro, R.C. Boucher. 2001. Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia. J. Gen. Physiol. 117: 53-67.
2. Coakley, R.D., A.M. Paradiso, B.R. Grubb, J.T. Gatzy, J.L, Chadburn, R.C. Boucher. 2000. Abnormal airway surface liquid Ph (pH_ASL) regulation in cultured CF bronchial epithelium. Ped. Pulmonol. Suppl. 20: 83.
3. Paradiso, A.M., C.M. Ribeiro, R.C. Boucher. 2000. Uridine triphosphates (UTP)-regulated Cl^- secretion is mediated by a Ca²⁺-independent protein kinase C (PKC). Ped. Pulmonol. Suppl. 20: 124.
4. Paradiso, A.M., L.H. Burch, L.G. Rochelle, S.M. Kreda, C.M Ribeiro, A. Winters, R.D. Coakley, R.C. Boucher. 2000. Functional identification and tissue distribution of two forms of H⁺, K⁺-ATPase proximal human airway. Ped. Pulmonol. Suppl. 20: 125.
5. Ribeiro, C.P., A.M. Paradiso, M. Carew, S. Shears, R.C. Boucher. 2000. Endoplasmic reticulum (ER) expression and agonist-sensitive calcium stores are increased in cystic fibrosis (CF) human airway epithelia. Ped. Pulmonol. Suppl 20: 202.
6. Paradiso, A.M., H.A. Brown, T.K. Harden, and R.C. Boucher. 1999. Heterogeneous responses of cell Ca²⁺ in human airway epithelium. Exp.Lung Res. 25:277-290.
7. Paradiso, A.M., R.C, Boucher. 1998. Identification of basolateral Cl^-/HCO₃^- exchange in human normal and cystic fibrosis airway epithelia. Ped. Pulmonol. Suppl. 17: 106.
8. Paradiso, A.M. 1997. ATP activated basolateral Na⁺/H⁺ exchange in human normal and cystic fibrosis airway epithelium. Am.J.Physiol. 273: L148-L158.
9. Lazarowski, E.R., A.M. Paradiso, W.C. Watt, T.K. Harden, and R.C. Boucher. 1997. UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y₂ receptor. Proc.Natl.Acad.Sci.U.S.A. 94: 2599-2603.
10. Van Scott, M.R., T.C. Chinet, A.D. Burnette, and A.M. Paradiso. 1995. Purinergic regulation of ion transport across nonciliated bronchiolar epithelial (Clara) cells. Am.J.Physiol. 269:L30-L37.
11. Stutts, M.J., E.R. Lazarowski, A.M. Paradiso, and R.C. Boucher. 1995. Activation of CFTR Cl^- conductance in polarized T84 cells by luminal extracellular ATP. Am.J.Physiol. 268:C425-C433.
12. Geary, C.A., C.W. Davis, A.M. Paradiso, and R.C. Boucher. 1995. Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency. Am.J.Physiol. 268:L1021-L1028.
13. Barker, P.M., K.K. Brigman, A.M. Paradiso, R.C. Boucher, and J.T. Gatzy. 1995. Cl^- secretion by trachea of CFTR(+/-) and (-/-) fetal mouse. Am. J. Respir. Cell Mol. Biol. 13:307-313.
14. Knowles, M.R., A.M. Paradiso, and R.C. Boucher. 1995. In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. Hum.Gene Ther. 6:445-455.
15. Paradiso, A.M., S.J. Mason, E.R. Lazarowski, and R.C. Boucher. 1995. Membrane-restricted regulation of Ca²⁺ release and influx in polarized epithelia. Nature 377:643-646.
16. Grubb, B.R., A.M. Paradiso, and R.C. Boucher. 1994. Anomalies in ion transport in CF mouse tracheal epithelium. Am.J.Physiol. 267:C293-C300.
17. Stutts, M.J., J.G. Fitz, A.M. Paradiso, and R.C. Boucher. 1994. Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP. Am.J.Physiol. 267:C1442-C1451.
18. Rhoads, J.M., W. Cheng, P. Chu, H.M. Berschneider, R.A. Argenzio, and A.M. Paradiso. 1994. L-glutamine and L-asparagine stimulate Na+/H+ exchange in a porcine jejunal enterocyte line. Am.J.Physiol. 266:G828-G838.
19. Parr, C.M., D.M. Sullivan, A.M. Paradiso, E.R. Lazarowski, L.H. Burch, J.C. Olsen, L. Erb, G.A. Weisman, R.C. Boucher, and J.T. Turner. 1994. Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc.Natl.Acad.Sci.U.S.A. 91:3275-3279
20. Clarke, L.L., A.M. Paradiso, S.J. Mason, and R.C. Boucher. 1992. Effects of bradykinin and Na+ and Cl- transport in human nasal epithelium. Am.J.Physiol. 262:C644-C655.
21. Clarke, L.L., A.M. Paradiso, and R.C. Boucher. 1992. Histamine induced chloride secretion in human nasal epithelium: coordinated response of apical and basolateral membranes. Am.J.Physiol. 263:1190-1199.
22. Van Scott, M.R. and A.M. Paradiso. 1992. Calcium-mediated regulation of ion transport in rabbit bronchiolar and tracheal epithelial cells. Am.J.Physiol. 263:L122-L127.
23. Paradiso, A.M. 1992. Identification of Na+/H+ exchange in human normal and cystic fibrosis ciliated airway epithelium. Am.J.Physiol. 262:L757-L764.
24. Noah, T.L., A.M. Paradiso, M.C. Madden, K.P. McKinnon, and R.B. Devlin. 1991. The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators. Am.J.Respir.Cell Mol.Biol. 103:484-492.
25. Paradiso, A.M., E.H.C. Cheng, and R.C. Boucher. 1991. Effects of bradykinin on intracellular calcium regulation in human ciliated airway epithelium. Am.J.Physiol. 261:L63-L69.