Convergence of Ca2+-desensitizing mechanisms activated by forskolin and phenylephrine pretreatment, but not 8-bromo-cGMP.
Contractile stimuli can sensitize myosin to Ca2+ by activating RhoA kinase (ROK) and PKC that inhibit myosin light chain phosphatase (MLCP) activity. Relaxant stimuli, acting through PKA and PKG (cyclic nucleotide-dependent protein kinases), and pretreatment with contractile agents such as phenylephrine (PE), can desensitize myosin to Ca2+. It is unknown precisely how these stimuli cause Ca2+ desensitization. To test the hypothesis that PKA, PKG, and PE pretreatment signaling systems converge to cause relaxation by inhibition of ROK in intact, isolated tissues, we examined the effects of forskolin (FSK; PKA activation), 8-bromo-cGMP (8br-cGMP; PKG activation), and PE pretreatment on KCl-induced force maintenance in rabbit arteries, a response nearly completely dependent on ROK activation. PE pretreatment and agents activating PKA and PKG caused Ca2+ desensitization by inhibiting KCl-induced tonic force and MLC phosphorylation without inhibiting intracellular [Ca2+]. At pCa 5 in beta-escin-permeabilized muscle, FSK and 8b-cGMP accelerated the relaxation rate when tissues were returned to pCa 9, suggesting that both agents can elevate MLCP activity. However, a component of the Ca2+ desensitization attributed to PKG activation in intact tissues appeared to involve a MLC phosphorylation-independent component. Inhibition of KCl-induced tonic force by the ROK inhibitor, Y-27632, and by PE pretreatment, were synergistically potentiated by 8b-cGMP, but not FSK. FSK and PE pretreatment, but not 8b-cGMP, inhibited the KCl-induced increase in site-specific myosin phosphatase target protein-1 phosphorylation at Thr853. These data support the hypothesis that PKA and PE pretreatment converge on a common Ca2+-desensitization pathway, but that PKG can act by a mechanism different from that activated by PKA and PE pretreatment.
Porter
Melissa
M
Virginia Commonwealth Univ. School of Medicine, Dept of Biochemistry, 1101 E. Marshall St., PO Box 980614, Richmond, VA 23298-0614, USA.
Evans
Melissa C
MC
Miner
Amy S
AS
Berg
Krystina M
KM
Ward
Kevin R
KR
Ratz
Paul H
PH
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000378:metabolism / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005263:Femoral Artery / Q000187:drug effects / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000187:drug effects / Q000378:metabolism; D010656:Phenylephrine / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D014662:Vasoconstrictor Agents / Q000378:metabolism / Q000494:pharmacology*
D014662:Vasoconstrictor Agents; D005576:Colforsin; D010656:Phenylephrine; C016276:8-bromocyclic GMP; D011189:Potassium Chloride; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D006152:Cyclic GMP; D002118:Calcium