Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.
Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.
Gil
Fulvio R
FR
Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St-Urbain St., Montréal, Québec, Canada H2X 2P2.
Zitouni
Nedjma B
NB
Azoulay
Eric
E
Maghni
Karim
K
Lauzon
Anne-Marie
AM
D000199:Actins / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D016535:Bronchial Hyperreactivity / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D016084:Bronchoconstriction / Q000187:drug effects / Q000502:physiology; D016085:Bronchoconstrictor Agents / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000235:genetics; D004195:Disease Models, Animal; D007536:Isomerism; D016210:Methacholine Chloride / Q000494:pharmacology; D009119:Muscle Contraction / Q000502:physiology; D009130:Muscle, Smooth / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011916:Rats, Inbred F344; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024745:Smooth Muscle Myosins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000199:Actins; D016085:Bronchoconstrictor Agents; D002148:Calmodulin-Binding Proteins; D018994:Myosin Light Chains; D016210:Methacholine Chloride; D024745:Smooth Muscle Myosins; D018995:Myosin Heavy Chains