title: Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus.
abstract: Ca(2+) 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 Ca(2+) 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/W(v) 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.
authors.affiliation: University of Nevada School of Medicine, Physiology and Cell Biology, University of Nevada, Reno, CMM203E, Reno, NV 89557, USA.
authors.forename: Bhupal P
authors.initials: BP
authors.lastname: Bhetwal
authors.forename: Kenton M
authors.initials: KM
authors.lastname: Sanders
authors.forename: Changlong
authors.initials: C
authors.lastname: An
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Trappanese
authors.forename: Robert S
authors.initials: RS
authors.lastname: Moreland
authors.forename: Brian A
authors.initials: BA
authors.lastname: Perrino
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002799:Cholinergic Fibers / Q000187:drug effects / Q000502:physiology; D004558:Electric Stimulation; D005748:Gastric Fundus / Q000294:innervation / Q000378:metabolism / Q000502:physiology*; D056885:Interstitial Cells of Cajal / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000294:innervation / Q000378:metabolism / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D009388:Neostigmine / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D009435:Synaptic Transmission
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D018727:Muscarinic Antagonists; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; C491520:Ppp1r14a protein, mouse; D047428:Protein Kinase Inhibitors; D009388:Neostigmine; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; C510645:Ppp1r12a protein, mouse; D002118:Calcium