PURINERGIC REGULATION OF ION-TRANSPORT ACROSS NONCILIATED BRONCHIOLAREPITHELIAL (CLARA) CELLS

Previous studies demonstrated that elevation of intracellular calcium concentration ([Ca2+](i)) increased electrogenic anion transport by br onchiolar epithelia. Extracellular nucleotides were shown to elevate [ Ca2+](i) and transepithelial short-circuit current (I-sc) in proximal airways epithelia. In this study purine and pyrimidine nucleotides wer e investigated for their ability to regulate ion transport by rabbit n onciliated bronchiolar epithelial (Clara) cells in culture. ATP in the apical bath induced a concentration-dependent transient increase in [ Ca2+](i) and I-sc. Mean effective doses (ED(50)) Of the responses were 10(-7) M and 10(-6) M, respectively. Transepithelial resistance (R(t) ) decreased. The peak changes in I-sc and R(t) were 7.8 +/- 1.2 mu A/c m(2) and -59 +/- 14 Omega . cm(2) (n = 26, basal I-sc = 47.4 +/- 4.3 m u A/cm(2) and R(t) = 428 +/- 40 Omega . cm(2)). Some preparations exhi bited a small residual increase in I-sc after the initial response, bu t the change was not statistically significant (Delta I-sc = 1.7 +/- 1 .2 mu A/cm(2), n = 18). Addition of ATP to the basolateral bath had no detectable effects. Purinoceptor agonists were used to characterize t he receptors mediating the change in I-sc. UTP and ATP gamma S increas ed I-sc and inhibited subsequent stimulation by ATP. ADP, ADP beta S, 2-methylthio-ATP, and alpha,beta-methylene-ATP had negligible effects on the peak Delta I-sc and subsequent stimulation by ATP. The ionic me chanism underlying the ATP-induced increase in I-sc was investigated w ith the use of specific ion-transport inhibitors and by ion substituti on. The peak Delta I-sc was not inhibited by amiloride in the apical b ath, bumetanide in the basolateral bath, bilateral Cl--free Krebs Ring er bicarbonate, or bilateral HCO3-/CO2-free Ringer. Substitution of N- methyl-D-glucamine for Na+ in the basolateral bathing solution and bil ateral substitution of gluconate for both Cl- and HCO3- inhibited the peak Delta I-sc. When preparations were pretreated with a combination of bilateral HCO3-/CO2-free Ringer and amiloride in the apical bath to unmask Cl- secretion, ATP induced a large increase in I-sc that was i nhibited by bumetanide in the basolateral bath. These results indicate d that activation of P-2U purinoceptors in the apical membrane of Clar a cells mobilized [Ca2+](i) and stimulated electrogenic secretion of C l- and HCO3-.