Differences in the protein-kinase-A-dependent regulation of CFTR Cl - channels and Na + -K + pumps in guinea-pig ventricular myocytes

Abstract

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

“…ϩ -K ϩ pump inhibition mediated by cAMP-dependent pathways in cardiac myocytes was reported in early studies (8,9), but many more recent studies have reported that these pathways mediate pump stimulation either by increasing maximal pump rate or by increasing pump affinity for intracellular Na ϩ (7,(11)(12)(13)(14)(15)(16)30). In our study, forskolin induced glutathionylation of the Na ϩ -K ϩ pump ␤ 1 subunit.…”

“…We have found the ␤ 3 adrenergic receptor mediates stimulation of the Na ϩ -K ϩ pump in cardiac myocytes (5). 5 We, therefore, used forskolin, as have most of the recent studies (7,11,12,15,16).…”

Dysfunction of the non-neuronal cholinergic system in the airways and blood cells of patients with cystic fibrosis

“…CFTR is a widely expressed transport protein and has been found outside the lung in sweat glands, uterine cervix, vas deferens, intestine, liver, pancreas, immune cells and heart (Davis et al, 1996;Kockskämper et al, 2001). CFTR is a member of the ABC transporter proteins and is involved in the regulation of the transport of monovalent ions, acetylcholine representing a monovalent permanent ion.…”

Blockade of nicotinic and muscarinic receptors facilitates spontaneous migration of human peripheral granulocytes: Failure in cystic fibrosis

“…CFTR is a widely expressed transport protein and has been found outside the lung in sweat glands, uterine cervix, vas deferens, intestine, liver, pancreas, immune cells and heart (Davis et al, 1996;Kockskämper et al, 2001). Previously it has been shown that the content of acetylcholine is reduced in isolated airways and peripheral leucocytes obtained from CF-patients and first evidence for a co-localization of choline-acetyltransferase (ChAT) and CFTR was found in human mononuclear cells .…”