CA2+ AS A 2ND MESSENGER IN CFPAC-1 CELLS

cAMP-stimulated Cl- secretion is known to be defective in patients with cystic fibrosis (CF). However, Ca2+ mediated electrolyte secretion still remains intact. We measured the intracellular Ca2+ activity ([Ca2+]i) in the permanent cystic fibrosis cell line CFPAC-1 in connection with electrophysiological investigations. Cells grown on glass coverslips for 2-7 days were loaded with fura-2. The influence of various known pancreatic secretagogues and agonists on [Ca2+]i were tested in an inverted fluorescence microscope. Application of ATP (10(-5) mol/1) raised [Ca2+]i within seconds from a mean resting value of 123 +/- 33 nmol/l to a peak value of 1,138 +/- 304 nmol/l (n = 13). [Ca2+]i returned to basal values when ATP was removed. Similarly, carbachol (CCH, 10(-5) mol/l; n = 14), neurotensin (NT, 10(-8) mol/l; n = 15), epinephrine (EPI, 10(-4) mol/l, n = 8) and vasoactive intestinal peptide (VIP 10(-8) mol/l, n = 6) increased [Ca2+]i (in nmol/l) from 101 +/- 24 to 386 +/- 62 (CCH), from 91 +/- 19 to 681 +/- 85 (NT), from 47 +/- 7 to 171 +/- 29 (EPI) and from 43 +/- 9 to 298 +/- 153 (VIP). The Ca2+ response was biphasic in the case of ATP and CCH with an initial peak declining to a plateau in the presence of the agonist. The EPI-induced [Ca2+]i increase was always transient, lasting approximately 60 s. Similarly, the VIP- and NT-induced [Ca2+]i increases were transient at concentrations > 10(-9) mol/l and showed a [Ca2+]i plateau at 10(-9) and 10(-10) (VIP) and 10(-9) (NT) mol/l. Using Ca2+-free bath solutions, each agonist still showed a transient [Ca2+]i increase. Hypotonic cell swelling (in mosm/l: 240; n = 5; 225; n = 7; 190; n = 6; 160; n = 13) induced an elevation of [Ca2+]i persisting as long as the hypotonic solution was present. The present data indicate that CFPAC-1 cells express P2, muscarinic, NT, VIP and alpha-receptors, all inducing a rise in [Ca2+]i. The [Ca2+]i elevation caused by these agonists and hypotonicity is probably responsible for the marked increase of Cl- and K+ conductances.