Complex Phosphatase Regulation of Ca2+-activated Cl- Currents in Pulmonary Arterial Smooth Muscle Cells

The present study was undertaken to determine whether the two ubiquitously expressed Ca2+-independent phosphatases PP1 and PP2A regulate Ca2+-activated Cl- currents (I-Cl(Ca)) elicited by 500 nM [Ca2+](i) in rabbit pulmonary artery (PA) myocytes dialyzed with or without 3 mm ATP. Reverse transcription-PCR experiments revealed the expression of PP1 alpha, PP1 beta/delta, PP1 gamma, PP2A alpha, PP2A beta, PP2B alpha (calcineurin (CaN) A alpha), and PP2B beta (CaN A beta) but not PP2B gamma (CaN A gamma) in rabbit PA. Western blot and immunofluorescence experiments confirmed the presence of all three PP1 isoforms and PP2A. Intracellular dialysis with a peptide inhibitor of calcineurin (CaN-AIP); the nonselective PP1/PP2A inhibitors okadaic acid (0.5, 10, or 30 nM), calyculin A (10 nM), or cantharidin (100 nM); and the selective PP1 inhibitor NIPP-1 (100 pM) potently antagonized the recovery of I-Cl(Ca) in cells dialyzed with no ATP, whereas the PP2A-selective antagonist fostriecin (30 or 150 nM) was ineffective. The combined application of okadaic acid (10 nM) and CaN-autoinhibitory peptide (50 mu M) did not potentiate the response of I-Cl(Ca) in 0 ATP produced by maximally inhibiting CaN or PP1/PP2A alone. Consistent with the non-additive effects of either classes of phosphatases, the PP1 inhibitor NIPP-1 (100 pM) antagonized the recovery of I-Cl(Ca) induced by exogenous CaN A alpha (0.5 mu M). These results demonstrate that ICl( Ca) in PA myocytes is regulated by CaN and PP1 and/or PP2A. Our data also suggest the existence of a functional link between these two classes of phosphatases.