Vasopressin and protein kinase A activate G protein-sensitive epithelial Na+ channels

1993 ◽  
Vol 265 (1) ◽  
pp. C218-C223 ◽  
Author(s):  
A. G. Prat ◽  
D. A. Ausiello ◽  
H. F. Cantiello
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
G Protein ◽  
Channel Number ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Intact Cells ◽  
Inside Out ◽  
Kinase A

To determine the molecular steps involved in the vasopressin-induced renal Na+ reabsorption, the patch-clamp technique was utilized to study the role of this hormone in the regulation of apical Na+ channels in renal epithelial A6 cells. Addition of arginine vasopressin (AVP) induced and/or enhanced Na+ channel activity within 5 min of addition under cell-attached conditions. The AVP-induced channel activity was a reflection of both an increase in the average apparent channel number (0.2-1.7) and the percent open time (2-56%). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, the adenosine 3',5'-cyclic monophosphate (cAMP) analogues, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, or forskolin elicited a comparable effect to that of AVP. The induced channels had similar properties to Na+ channels previously reported, including a channel conductance of 9 pS, Na(+)-to-K+ selectivity of 3-5:1, and high amiloride sensitivity. The cAMP-dependent protein kinase A (PKA) in the presence of ATP induced and/or enhanced Na+ channel activity in excised inside-out patches with a change in average apparent channel number and percent open probability similar to those observed with either AVP or cAMP analogues in intact cells. Addition of activated pertussis toxin (100 ng/ml) completely blocked the AVP- or PKA-induced Na+ channel activity in excised inside-out patches, whereas incubation of intact cells with the toxin completely prevented the effect of both activators. The data indicate that AVP mediates its effect through a cAMP-dependent pathway involving PKA activation whose target is the G protein pathway that regulates apical epithelial Na+ channel activity.

Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers

1997 ◽  
Vol 272 (4) ◽  
pp. C1262-C1270 ◽  
Author(s):  
B. K. Berdiev ◽  
V. G. Shlyonsky ◽  
O. Senyk ◽  
D. Keeton ◽  
Y. Guo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Lipid Bilayers ◽  
Na Channel ◽  
Type Ii ◽  
Na Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Atii Cell ◽  
Kinase A

Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.

Activation of epithelial Na+ channels by protein kinase A requires actin filaments

1993 ◽  
Vol 265 (1) ◽  
pp. C224-C233 ◽  
Author(s):  
A. G. Prat ◽  
A. M. Bertorello ◽  
D. A. Ausiello ◽  
H. F. Cantiello
Keyword(s):  
Protein Kinase ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activation ◽  
Epithelial Na Channels ◽  
Inside Out ◽  
Kinase A

We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated actin filaments induced Na+ channel activity in excised inside-out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 degrees C. These data suggest that the form of actin required for Na+ channel activation (i.e., "short" actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate the activation of Na+ channels by PKA.

scholarly journals Protein Kinase A-mediated Phosphorylation of the Gα13Switch I Region Alters the Gαβγ13-G Protein-coupled Receptor Complex and Inhibits Rho Activation

2002 ◽  
Vol 278 (1) ◽  
pp. 124-130 ◽  
Author(s):  
Jeanne M. Manganello ◽  
Jin-Sheng Huang ◽  
Tohru Kozasa ◽  
Tatyana A. Voyno-Yasenetskaya ◽  
Guy C. Le Breton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
G Protein ◽  
Receptor Complex ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Kinase A

Phosducin is a protein kinase A-regulated G-protein regulator

Nature ◽  
1992 ◽  
Vol 358 (6381) ◽  
pp. 73-76 ◽  
Author(s):  
Petra H. Bauer ◽  
Stefan Müller ◽  
Mechthild Puzicha ◽  
Susanne Pippig ◽  
Brigitte Obermaier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
G Protein ◽  
Kinase A

The heterotrimeric G‐protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides

2020 ◽  
Vol 22 (10) ◽  
Author(s):  
Marco Iván Valle‐Maldonado ◽  
José Alberto Patiño‐Medina ◽  
Carlos Pérez‐Arques ◽  
Nancy Yadira Reyes‐Mares ◽  
Irvin Eduardo Jácome‐Galarza ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
G Protein ◽  
Hyphal Growth ◽  
Mucor Circinelloides ◽  
Beta Subunit ◽  
Low Oxygen ◽  
Heterotrimeric G Protein ◽  
Oxygen Conditions ◽  
Kinase A

Ca2+-, phorbol ester-, and cAMP-stimulated enzyme secretion from permeabilized rat pancreatic acini

1986 ◽  
Vol 250 (5) ◽  
pp. G698-G708 ◽  
Author(s):  
T. Kimura ◽  
K. Imamura ◽  
L. Eckhardt ◽  
I. Schulz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Release ◽  
Enzyme Secretion ◽  
Dependent Manner ◽  
Intact Cells ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Kinase A

Enzyme secretion from the exocrine pancreas is stimulated by receptor-activated breakdown of phosphatidylinositol 4,5-bisphosphate and consequent rise of both inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which leads to Ca2+ release and to activation of protein kinase C, respectively. Another way involves receptor-mediated stimulation of adenylate cyclase and consequent rise of cAMP and activation of protein kinase A. In the present work we have studied direct stimulation, inhibition, and mutual interaction of these pathways on enzyme secretion from isolated rat pancreatic acini that had been permeabilized by treatment with saponin or digitonin. The data were compared with those obtained in isolated intact acini. The data show that with increasing free Ca2+ concentrations greater than 10(-6) M protein release increases in "leaky" but not in "intact" cells and is maximal at approximately 10(-3) M, increasing about twofold compared with that in the absence of Ca2+. In the presence of the acetylcholine analogue carbachol, this effect of Ca2+ is enhanced by about threefold in leaky cells and is also present in intact cells to a similar extent. cAMP and its analogues, dibutyryl cAMP (dbcAMP) and 8-bromo-cAMP stimulate protein release by about twofold in the presence of Ca2+ in leaky cells. In intact acini cAMP has no effect, and cAMP analogues stimulate enzyme secretion by about twofold in some but not all experiments. Similarly, forskolin, an activator of adenylate cyclases and inhibitors of cyclic nucleotide-dependent phosphodiesterases, such as 3-isobutyl-1-methylxanthine (IBMX) and R0 201724, stimulate protein release in permeabilized acini. The Ca2+-binding protein calmodulin has no effect on enzyme secretion, whereas the calmodulin antagonist trifluoperazine dihydrochloride stimulates protein release in leaky but not in intact acini. The activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates protein release in a Ca2+-dependent manner and enhances cAMP-induced secretion. The effects of carbachol, TPA, cAMP, and a combination of both TPA and cAMP are inhibited by the polyamine spermine in permeabilized cells. Spermine has no effect on carbachol-induced enzyme secretion in intact cells. The data suggest that enzyme secretion from pancreatic acinar cells is mediated by cAMP protein kinase A and by Ca2+ phospholipid protein kinase C in a Ca2+-dependent way and that interaction occurs between both pathways.

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