Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus

• Published in: The Journal of physiology Vol. 591; no. 12; pp. 2971 - 2986
• Main Authors: Bhetwal, Bhupal P., Sanders, Kenton M., An, Changlong, Trappanese, Danielle M., Moreland, Robert S., Perrino, Brian A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 15-06-2013; Wiley Subscription Services, Inc; Blackwell Science Inc
• Subjects: Animals; Calcium - metabolism; Cholinergic Fibers - drug effects; Cholinergic Fibers - physiology; Electric Stimulation; Gastric Fundus - innervation; Gastric Fundus - metabolism; Gastric Fundus - physiology; Interstitial Cells of Cajal - physiology; Kinases; Male; Mice; Mice, Inbred C57BL; Molecular and Cellular; Muscarinic Antagonists - pharmacology; Muscle Contraction; Muscle Proteins - metabolism; Muscle, Smooth - innervation; Muscle, Smooth - metabolism; Muscle, Smooth - physiology; Myosin Light Chains - metabolism; Myosin-Light-Chain Kinase - metabolism; Myosin-Light-Chain Phosphatase; Neostigmine - pharmacology; Phosphatase; Phosphoproteins - metabolism; Phosphorylation; Protein Kinase Inhibitors - pharmacology; Smooth muscle; Synaptic Transmission
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2013.255745

Key points
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In smooth muscles, the sensitivity of contraction to Ca2+ can be increased by the phosphorylation of CPI‐17 and MYPT1, resulting in the inhibition of myosin light chain phosphatase (MLCP).
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Ca2+ sensitization of smooth muscle contraction has typically been studied by immersing muscles in solutions containing contractile agonists.
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However, stimulating muscles by bath‐applied agonists may not be equivalent to neurotransmitter release because different post‐junctional receptors may be activated in response to these different modes of stimulation.
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In this study we found that a bath‐applied cholinergic agonist activates Ca2+ sensitization mechanisms in gastric fundus smooth muscles that are different than those of cholinergic neurotransmission. Electrical field stimulation (EFS) only increased CPI‐17 phosphorylation, while bath‐applied carbachol increased both CPI‐17 and MYPT1 phosphorylation.
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With the cholinesterase inhibitor neostigmine present, both CPI‐17 and MYPT1 phosphorylation were increased by EFS.
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In fundus muscles of W/Wv mice which lack intramuscular interstitial cells of Cajal (ICC‐IMs), EFS alone increased both CPI‐17 and MYPT1 phosphorylation.
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These findings indicate that ACh availability determines which Ca2+ sensitization mechanisms are activated, and ICC‐IMs regulate the access of ACh to smooth muscles.
  Ca2+ sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath‐applied agonists and enteric neurotransmission activate similar Ca2+ sensitization mechanisms. We investigated protein kinase C (PKC)‐potentiated phosphatase inhibitor protein of 17 kDa (CPI‐17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath‐applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI‐17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/Wv mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC‐dependent CPI‐17 phosphorylation. Bath‐applied CCh recruits additional ROCK‐dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC‐IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC‐IMs. These results provide the first biochemical evidence for focalized (or synaptic‐like) neurotransmission, rather than diffuse ‘volume’ neurotransmission in a smooth muscle tissue. Furthermore, these findings demonstrate that bath application of contractile agonists to gastrointestinal smooth muscles does not mimic physiological responses to cholinergic neurotransmission.