Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2009; 284(23):15541-8. DOI: 10.1074/jbc.M900888200
Source: PubMed


Relationships among biochemical signaling processes involved in Ca2+/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase
(MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca2+-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations
of the relationships among [Ca2+]i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the
onset of [Ca2+]i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas
force latency was 109 ± 3 ms. [Ca2+]i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal
value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated
with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca2+]i and also declined more rapidly than [Ca2+]i during relaxation. The apparent desensitization of MLCK to Ca2+ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain
phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for
force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in
[Ca2+]i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca2+ signaling complex as an elementary mechanism initiating contraction.

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    • "Serine 1760 is located in the calmodulin binding site in the C-terminal domain of smMLCK. Phosphorylation at this site prevents calcium-calmodulin complex from interacting with smMLCK and activating smMLCK [58]. Whether IgE regulates the activation of smMLCK and subsequent phosphorylation of myosin RLC and contributes directly in HASM cells contraction, would be interesting to investigate. "
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    ABSTRACT: Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.
    PLoS ONE 04/2014; 9(4):e93946. DOI:10.1371/journal.pone.0093946 · 3.23 Impact Factor
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    • "In contrast to results obtained with CPI - 17 , our data did not support a role for MYPT1 phosphorylation in mediating Ca 2+ sensitization of the contractile response evoked by EFS . We observed a small increase in phosphorylation of MYPT1 - Thr850 at 50 Hz , which is consistent with previous data ( Ding et al . 2009 ) . Under resting conditions , MYPT1 is phosphorylated at both Thr696 and Thr850 . Thus , a constitutively active ROCK may contribute to basal MYPT1 phosphorylation in mouse bladder smooth muscle as evidenced by other studies ( Hashitani et al . 2004 ; Poley et al . 2008 ) . We earlier provided evidence showing differences in sensitivit"
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    ABSTRACT: Key points • Parasympathetic nerves release the neurotransmitters ATP and acetylcholine that activate purinergic and muscarinic receptors, respectively, to initiate contraction of urinary bladder smooth muscle. • Although both receptors mediate Ca(2+) influx for myosin regulatory light chain (RLC) phosphorylation necessary for contraction, the muscarinic receptor may also recruit cellular mechanisms affecting the Ca(2+) sensitivity of RLC phosphorylation. • Using transgenic mice expressing Ca(2+)/calmodulin sensor myosin light chain kinase (MLCK) in smooth muscles, the effects of selective purinergic or muscarinic receptor inhibition were examined on neurally stimulated tissues in relation to signalling pathways converging on RLC phosphorylation. • Purinergic-mediated Ca(2+) signals provide the initial Ca(2+)/calmodulin activation of MLCK with muscarinic receptors supporting sustained responses. • Activation of muscarinic receptors leads phosphorylation of myosin light chain phosphatase inhibitor CPR-17 to enhance Ca(2+) sensitivity while also initiating phosphorylation-dependent Ca(2+) desensitization of MLCK. The interplay between Ca(2+) sensitization and desensitization mechanisms fine tunes the contractile signalling module for force development.
    The Journal of Physiology 08/2012; 590(Pt 20):5107-21. DOI:10.1113/jphysiol.2012.235424 · 5.04 Impact Factor
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    • "Contraction follows the release of neurotransmitters from motor nerves that are dispersed in the muscle bundles [3]. This results in a rapid elevation of the sarcoplasmic Ca2+ concentration, activation of myosin light chain kinase, phosphorylation of myosin, and, after a brief delay, force development [4]. Acetylcholine is central among the transmitters released from neural varicosities in the detrusor, and the muscarinic Gq-coupled M3 receptor is primarily responsible for cholinergic detrusor activation [5]. "
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    ABSTRACT: MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+) channels in the detrusor.
    PLoS ONE 04/2012; 7(4):e35882. DOI:10.1371/journal.pone.0035882 · 3.23 Impact Factor
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