As detailed in the methods, two antibodies were made to C-terminal PLM peptides. To evaluate the specificity of these antibodies, purified recombinant PLM [29
] (shown to be dephosphorylated by mass spectrometry) was phosphorylated with the catalytic subunit of PKA in the presence of γ-32
P-ATP. Different amounts of phosphorylated and dephosphorylated PLM (exposed to γ-32
P-ATP without PKA) were loaded on SDS gels, blotted, analyzed for 32
P activity, and immunoblotted with both antibodies
. Increasing 32
P activity was observed with increasing amount of PKA-treated PLM (, top panel). Calculated stoichiometry revealed that ~50% of PLM was phosphorylated by PKA treatment. Dephosphorylated PLM was not detected by the CP68 antibody
(, second panel). The intensity of the CP68 antibody signal was proportional to the amount of PKA treated PLM loaded on the immunoblot
(, third panel). The intensity of the C2 antibody signal was proportional to the amount of dephosphorylated PLM loaded on the immunoblot
(, fourth panel). The intensity of the C2 antibody signal was less intense on immunoblots loaded with PKA-treated PLM
(, bottom panel) compared to
that observed with dephosphorylated PLM.
Specificity of the anti-PLM antibodies.
Swine carotid medial smooth muscle rings were first equilibrated and then contracted with 10 μM histamine or 40 mM [K+]o for 10 min. Some were then relaxed with 0.1, 0.3, 1, 3, or 10 μM forskolin, and others were left in the contraction solution. After an additional 30 min, tissues were frozen and homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots. As detailed in the methods, the intensity of both the C2 and CP68 antibodies were normalized to internal standards. For clarity the term “C2 signal” will refer to the intensity the C2 antibody immunoreactivity normalized to the intensity of C2 antibody immunoreactivity from the untreated control tissue homogenate loaded on the same blot. Similarly, the term “CP68 signal will refer to the intensity of the CP68 antibody immunoreactivity normalized to the intensity of the CP68 antibody immunoreactivity from the KF pooled homogenate loaded on the same blot. This normalization procedure allows comparison of changes in the C2 and CP68 antibody intensity on different immunoblots. shows a representative immunoblot of tissues stimulated with histamine, and shows aggregate data. PLM was present in swine arterial smooth muscle, and its phosphorylation status depended on the treatment.
Changes in the CP68 (top panel) and C2 (bottom panel) PLM signals in swine carotid artery tissue homogenates.
Unstimulated (control) tissues had low levels of CP68 signal and force (lane 2 in and the filled symbols in ). 10 μM histamine induced a large decrease in the C2 signal, a small increase in the CP68 signal, and a maximal contraction (lane 3 in and open squares in ). Adding forskolin to histamine stimulated tissues was associated with no change in the C2 signal, dose dependent increases in the CP68 signal (lanes 4–7 in ), and dose dependent reduction in force ().
40 mM [K+]o depolarization induced a maximal contraction without significant changes in the C2 or CP68 signal (open circles in ). Adding forskolin to high [K+]o depolarized tissues was associated with a reduced C2 signal, dose dependent increases in the CP68 signal, and dose dependent reduction in force. When relaxed with forskolin, the histamine stimulated tissues exhibited a greater CP68 signal and lower force than that observed in the 40 mM [K+]o-depolarized tissues.
The relation between the CP68/KF ratio and active force is shown in . There was a highly significant negative correlation between the CP68 signal and force (r2 = 0.5, p<0.0001 ANCOVA). There was no significant difference in the slopes or intercept of the regression lines for tissues stimulated with histamine or high [K+]o, suggesting a simple inverse relation between the degree of forskolin-induced relaxation and the degree of serine 68 PLM phosphorylation.
We evaluated whether agents that increase [cGMP] also induce PLM phosphorylation. 10 μM histamine induced a small increase in the CP68 signal and a maximal contraction. Addition of 100 μM nitroglycerin (to increase [cGMP]) induced a relaxation without changing the CP68 signal from the values observed with histamine alone. Addition of 1 μM forskolin (to increase [cAMP]) further increased the CP68 signal.
If S68 PLM phosphorylation were to cause relaxation by increasing the activity of the Na,K ATPase, then forskolin should reduce intracellular [Na+
is difficult to measure in intact smooth muscle, but can be inferred by the response to removal of extracellular [Na+
]. If L-type Ca2+
channels are blocked, contraction induced by removal of [Na+
should result from the increase in [Ca2+
that occurs from Ca2+
influx through reversal of the Na-Ca exchanger. If forskolin were to reduce [Na+
, then there would be less Na+ for the Na-Ca exchanger, less increase in [Ca2+
, and less contraction. We evaluated the effect of forskolin pretreatment on swine carotid artery contraction induced by a zero Na+
saline in the presence of 10 μM diltiazem (). Pretreatment with 1 μM forskolin for 15 min significantly reduced the zero Na+
contraction compared to a control that was not treated with forskolin.
Forskolin dependent contractile response to removal of extracellular [Na+].