The outwardly rectifying chloride channel in rat peritoneal mast cells is regulated by serine/threonine kinases and phosphatases.
A slowly activating, outwardly rectifying Cl- channel (ORCC) has been described in rat peritoneal mast cells (RPMCs). This channel is activated by intracellular application of cAMP, an effect that might be mediated by a PKA-type serine/threonine protein kinase. To test this hypothesis, whole-cell patch-clamp experiments (nystatin-perforated patch) were performed and 8-bromoadenosine 3',5'-cyclic monophosphothioate, Sp-enantiomer (Sp-8Br-cAMPS), a cell membrane-permeable activator of PKA, and three inhibitors of different serine/threonine protein phosphatases (okadaic acid, cantharidin, calyculin A), were tested. In RPMCs application of repetitive series of step hyper- and depolarizations (holding potential 0 mV, test potentials -80 to +80 mV, step size +20 mV) induced a slowly increasing, [half-maximal activation time ( t0.5) 11.0+/-1.1 min, Imax (at +80 mV) 18.7+/-3.1 pA pF-1], DIDS-sensitive, outwardly rectifying Cl- current I(Cl,OR). The activation of this current could be accelerated by Sp-8Br-cAMPS, okadaic acid or cantharidin in the extracellular solution. Co-application of Sp-8Br-cAMPS and okadaic acid increased Imax supra-additively. Calyculin A and higher concentrations of cantharidin inhibited the Cl- current via unknown mechanisms. Our findings suggest that I(Cl,OR) in RPMCs is activated by a PKA-type protein kinase, a process which is antagonized functionally by okadaic acid- and cantharidin-sensitive protein phosphatases.
Seebeck
Jörg
J
Department of Pharmacology, University of Kiel, Hospitalstrasse 4, 24105 Kiel, Germany. jseebeck@pharmakologie.uni-kiel.de
Tritschler
Stefan
S
Roloff
Tim
T
Kruse
Marie-Luise
ML
Schmidt
Wolfgang E
WE
Ziegler
Albrecht
A
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D008387:Marine Toxins; D008407:Mast Cells / Q000201:enzymology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010529:Peritoneal Cavity / Q000166:cytology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013873:Thionucleotides / Q000494:pharmacology
C105593:8-bromoadenosine-3',5'-cyclic monophosphorothioate; D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D013873:Thionucleotides; D019319:Okadaic Acid; D015124:8-Bromo Cyclic Adenosine Monophosphate; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases