Kate Bárány

Exchange of the actin-bound nucleotide in intact arterial smooth muscle.

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In 32P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [γ,β-32P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the β-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the γ-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K+, as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.

Absence of calponin phosphorylation in contracting or resting arterial smooth muscle.

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 256, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 33P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [33P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.

Myosin light chain isoforms and their phosphorylation in arterial smooth muscle.

Arterial smooth muscle myosin contains nonphosphorylated and phosphorylated light chains that appear as 4 spots on two-dimensional, Coomassie blue-stained gel electrophoretograms at the 20,000-molecular weight level (referred to as spots 4 through 1 in order of decreasing isoelectric points). Anti-light chain recognizes the proteins in all 4 light chain spots. Complete dephosphorylation of light chain in muscle homogenate, by inhibiting myosin light chain kinase and by adding phosphatase, leads to 2 spots on two-dimensional gel electrophoretograms; both spots are visible on immunoblots. Stimulation (K+ or stretch) of smooth muscle results in increased light chain phosphorylation. Autoradiography of the gel electrophoretograms reveals that radioactive components are contained in spots 3, 2, 1, and in an additional spot with lower isoelectric point, referred to as spot 0. Phosphoamino acid analysis shows that spots 3 and 1 contain phosphoserine, whereas spots 2 and 0 contain phosphoserine and phosphothreonine. Two-dimensional phosphopeptide mapping of the trypsin-digested proteins from spots 3 and 1 shows predominantly 2 peptides; whereas from spots 2 and 0, it shows 5 peptides. Sodium dodecyl sulfate gel electrophoresis of the phosphopeptides obtained with Staphylococcus aureus V8 digestion gives identical maps for spots 3 and 2, which are different from the identical maps of spots 1 and 0. The results suggest that arterial smooth muscle myosin contains 2 nonphosphorylated 20,000-dalton light chain isoforms with different amino acid sequences and that each isoform can be mono- and diphosphorylated.

Calponin phosphorylation does not accompany contraction of various smooth muscles

Calponin phosphorylation was quantitated in 32P-labeled porcine arterial, uterine, tracheal, stomach and bladder smooth muscles. The negligible amount of 0.003-0.008 mol [32P]phosphate/mol calponin in resting muscles did not increase upon contraction induced by various agents, under conditions when myosin light-chain phosphorylation increased several-fold. The results indicate no involvement of calponin phosphorylation is smooth-muscle contraction.

Effect of okadaic acid on phosphorylation-dephosphorylation of myosin light chain in aortic smooth muscle homogenate

Myosin light chain phosphorylation in aortic smooth muscle homogenate reached a maximal level of 0.75 mol phosphate/mol light chain, and then declined. Addition of okadaic acid led to a sustained phosphorylation level of 1.7 mol/mol. In the absence of okadaic acid, phosphorylation was predominantly due to myosin light chain kinase, whereas in the presence of okadaic acid both myosin light chain kinase and protein kinase C were involved in phosphorylation. Okadaic acid inhibited dephosphorylation of the distinct sites in LC phosphorylated by either myosin light chain kinase or protein kinase C, suggesting that it exerts its effect through inhibition of myosin light chain phosphatases present in aortic homogenate.

Involvement of calponin and caldesmon in sustained contraction of arterial smooth muscle

The molecular mechanism of smooth muscle contraction was approached by a novel method, covalent 14C-labeling. Intra- and intermolecular protein interactions during contractile activity are reflected by changed reactivity of protein side chains; these can be detected by reagents which readily permeate through the muscle membrane without affecting the contractility and form covalent bonds with proteins in the muscle. The incorporation of 14CH2ICONH2 into proteins of 1-hour histamine contracted versus resting porcine carotid arterial muscles was determined. Out of fourteen 14C-labeled proteins analyzed, only two showed a change in reactivity during sustained contraction. The incorporation of 14CH2ICONH2 into calponin and caldesmon in contracted muscles was about 66% of that into these same proteins in resting muscles. A transformation of calponin and caldesmon molecules from an extended to a more compact conformation explains the decreased reactivity.

Protein phosphorylation during the contraction-relaxation-contraction cycle of arterial smooth muscle

Porcine carotid arterial muscles were labeled with 32P and then subjected to a resting-contraction-relaxation-contraction cycle. Four different agents were used for contraction: KCl, histamine, norepinephrine, and phorbol dibutyrate. To relax the contracted muscles, they were washed with physiological salt solution. Changes in the [32P]phosphate content of four different proteins--myosin light chain, a 28-kDa cytosolic protein, desmin, and caldesmon--were followed. In a short contraction-relaxation-contraction cycle lasting minutes, induced by K+, histamine, or norepinephrine, only the light chain underwent a phosphorylation-dephosphorylation-rephosphorylation without concomitant cyclic phosphorylation of the 28-kDa protein, desmin, or caldesmon. In a contraction-relaxation-contraction cycle of long duration, 60-min contractions with K+, histamine, or norepinephrine, cyclic phosphorylation of both the light chain and desmin was observed. With 60-min phorbol dibutyrate stimulation, in the long contraction-relaxation-contraction cycle, the phosphorylations of the light chain, desmin, and caldesmon were cycling. It is concluded that under physiological conditions, light-chain phosphorylation initiates both short and sustained arterial contraction. Desmin phosphorylation is likely to be involved in force maintenance during sustained contraction.

Characterization of the phosphorylatable myosin light chain in rat uterus

The 20 kDa myosin light chain of 32P-labeled rat uterus exhibited four spots on two-dimensional gel electrophoretograms; the corresponding autoradiograms revealed that three spots were radioactive. Completely dephosphorylated light chain exhibited three spots on electrophoretograms. Serine and threonine residues of the light chain were found to be phosphorylated in the uterus at a ratio of 6 to 1. During contraction, the amount of each phosphoamino acid increased proportionally to the increase in the total phosphate content of the light chain.

Stretch activates myosin light chain kinase in arterial smooth muscle

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.