Calponin is required for agonist-induced signal transduction--evidence from an antisense approach in ferret smooth muscle.
1. The present study was undertaken to determine whether calponin (CaP) participates in the regulation of vascular smooth muscle contraction and, if so, to investigate the mechanism. 2. By PCR homology cloning, the cDNA sequence of ferret basic (h1) CaP was determined and phosphorothioate antisense and random oligonucleotides were synthesized and introduced into strips of ferret aorta by a chemical loading procedure. 3. Treatment of ferret aorta with CaP antisense oligonucleotides resulted in a decrease in protein levels of CaP to 54% of that in random sequence-loaded muscles, but no change in the protein levels of caldesmon (CaD), actin, desmin or extracellular regulated protein kinase (ERK). 4. Contraction in response to phenylephrine or a phorbol ester was significantly decreased in antisense-treated muscles compared to random sequence-loaded controls. Neither basal intrinsic tone nor the contraction in response to 51 mM KCl was significantly affected by antisense treatment. 5. During phenylephrine contractions, phospho-ERK levels increased, as did myosin light chain (LC20) phosphorylation. Phenylephrine-induced ERK phosphorylation and CaD phosphorylation at an ERK site were significantly decreased by CaP antisense. Increases in myosin light chain phosphorylation were unaffected. 6. The data indicate that CaP plays a significant role in the regulation of contraction and suggest that in a tonically active smooth muscle CaP may function as a signalling protein to facilitate ERK-dependent signalling, but not as a direct regulator of actomyosin interactions at the myofilament level.
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H D
HD
Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Gangopadhyay
S S
SS
Ashworth
T D
TD
Morgan
K G
KG
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001011:Aorta; D001483:Base Sequence / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D005289:Ferrets; D008840:Microfilament Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009924:Organ Culture Techniques; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology*; D014661:Vasoconstriction / Q000502:physiology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D000096985:Calponins
D000316:Adrenergic alpha-Agonists; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D020928:Mitogen-Activated Protein Kinases; D018994:Myosin Light Chains; D016376:Oligonucleotides, Antisense; D010656:Phenylephrine; D014662:Vasoconstrictor Agents; D000096985:Calponins