Abstract
Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/W V mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/W V gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.
Highlights
Smooth muscle contraction and relaxation involves the phosphorylation and dephosphorylation of the 20kDa regulatory light chain of myosin (MLC) by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), respectively
The guanylyl cyclase (GC) inhibitor, ODQ (1μM) blocked the increase in telokin S13 phosphorylation by sodium nitroprusside (SNP), but had no effect on the increase in telokin S13 phosphorylation evoked by 8Br-cGMP (Fig 1B and 1C, n = 6)
We found that telokin S13 is basally phosphoryl-ated and that the exogenous cyclic GMP-elevating agents 8-Bromo-cGMP and SNP increased the basal telokin phosphorylation
Summary
Smooth muscle contraction and relaxation involves the phosphorylation and dephosphorylation of the 20kDa regulatory light chain of myosin (MLC) by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), respectively. Contraction is initiated by an increase in cytosolic Ca2+ ([Ca2+]i) and activation of Ca2+/CaM-dependent MLCK that phosphorylates MLC at S19. MLCP-dependent dephosphorylation of S19 moderates contractile force and eventually causes relaxation when [Ca2+]i is restored to resting levels. Contractile force is a function of the activity ratio between MLCK and MLCP. Decreasing the activity of MLCP leads to a phenomenon known as ‘Ca2+ sensitization’ in which a given increase in [Ca2+]i can yield a greater level of MLC phosphorylation and contractile force [1, 2]. Ca2+ sensitization occurs through regulatory proteins, such as CPI-17 and MYPT1, that when phosphorylated by protein kinase C (PKC) or Rho kinase (ROCK), inhibit MLCP [1, 3,4,5,6,7]
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