Serine 68 phospholemman phosphorylation during forskolin-induced swine carotid artery relaxation.
BACKGROUND
Phospholemman (PLM) is an abundant phosphoprotein in the plasma membrane of cardiac, skeletal and smooth muscle. It is a member of the FXYD family of proteins that bind to and regulate the Na,K-ATPase. Protein kinase A (PKA) is known to phosphorylate PLM on serine 68 (S68), although the functional effect of S68 PLM phosphorylation is unclear. We therefore evaluated S68 PLM phosphorylation in swine carotid arteries.


METHODS
Two anti-PLM antibodies, one to S68 phosphorylated PLM and one to unphosphorylated PLM, were made to PLM peptides in rabbits and tested with purified PLM and PKA-treated PLM. Swine carotid arteries were mounted isometrically, contracted, relaxed with forskolin and then homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots.


RESULTS
The antipeptide antibody 'C2' primarily reacted with unphosphorylated PLM, and the antipeptide antibody 'CP68' detected S68 PLM phosphorylation. Histamine stimulation of intact swine carotid artery induced a contraction, increased the CP68 PLM antibody signal and reduced the C2 PLM antibody signal. High extracellular [K(+)] depolarization induced a contraction without altering the C2 or CP68 PLM signal. Forskolin-induced relaxation of histamine or extracellular [K(+)] contracted arteries correlated with an increased CP68 signal. Nitroglycerin-induced relaxation was not associated with changes in the C2 or CP68 PLM signal.


CONCLUSIONS
These data suggest that a contractile agonist increased S68 PLM phosphorylation. Agents that increase [cAMP], but not agents that increase [cGMP], increased S68 PLM phosphorylation. S68 PLM phosphorylation may be involved in cAMP-dependent regulation of smooth muscle force.
Rembold
Christopher M
CM
Cardiovascular Division, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA 22908, USA. crembold@virginia.edu
Ripley
Marcia L
ML
Meeks
Melissa K
MK
Geddis
Lisa M
LM
Kutchai
Howard C
HC
Marassi
Francesca M
FM
Cheung
Joseph Y
JY
Moorman
J Randall
JR
D000818:Animals; D002339:Carotid Arteries / Q000187:drug effects* / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D009994:Osmolar Concentration; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine; D012964:Sodium / Q000378:metabolism; D013552:Swine; D014664:Vasodilation / Q000502:physiology*
D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D005576:Colforsin; D012694:Serine; D012964:Sodium; D000242:Cyclic AMP