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.

Figure 1. Effects of ‘bath-applied’ CCh on gastric fundus smooth muscle contraction and CPI-17 and MYPT1 phosphorylation

A, representative traces of CCh-evoked contraction of gastric fundus smooth muscles, showing the time points at which CPI-17 and MYPT1 phosphorylation were analysed. Mean ratios of the pT38/CPI-17 density values (±SD) (B) or the pT853/MYPT1 and pT696/MYPT1 density values (±SD) (C) and representative Western blots of pT38 (B), pT853 and pT696 (C) at each time point. n= 5, duplicate blots. *P < 0.05, #P < 0.01, §P < 0.001, compared to control (Con).

Figure 2. Effects of EFS on gastric fundus smooth muscle contraction and CPI-17 and MYPT1 phosphorylation

A, representative traces of contractions evoked by different frequencies of EFS showing the time points at which CPI-17 and MYPT1 phosphorylation were analysed. Mean ratios of the pT38/CPI-17 density values (±SD) (B), pT696/MYPT1 density values (±SD) (C), and pT853/MYPT1 density values (±SD) (D) and representative Western blots of pT38 (B), pT696 (C) and pT853 (D) at each time point. n= 5, duplicate blots. *P < 0.05, #P < 0.01, §P < 0.001, compared to control (white bar).

Figure 3. Pharmacological analysis of CCh-evoked CPI-17 and MYPT1 phosphorylation

Upper panels, mean ratios of the pT38/CPI-17 density values (±SD) (A, D, G and J) or the pT853/MYTP1 and pT696/MYPT1 density values (±SD) (B, E, H and K) in the absence or presence of CCh, or CCh in the presence of atropine (A and B), SAR1x (D and E), nicardipine (G and H), Go6976 or Gf109203x (J and K); lower panels, representative Western blots. Representative traces showing that atropine (C) blocks the CCh-evoked contraction, and SAR1x (F), nicardipine (I), Go6976 or Gf109203x (L) inhibit the CCh-evoked contraction. n= 5, duplicate blots. *P < 0.05, #P < 0.01, §P < 0.001 compared to control (white bars). †P < 0.05, ‡P < 0.01, ¶P < 0.001 compared to CCh.

Figure 4. Pharmacological analysis of CPI-17 phosphorylation evoked by EFS

Upper panels, mean ratios of the pT38/CPI-17 density values (±SD) without or with EFS, or EFS in the presence of atropine (A), nicardipine, Go6976, or Gf109203x (B); lower panels, representative Western blots. Representative traces showing that atropine (C) blocks the EFS-evoked contraction, and nicardipine, Go6976 or Gf109203x (D) inhibit the EFS-evoked contraction. n= 5, duplicate blots, *P < 0.05, #P < 0.01, §P < 0.001 compared to control (white bars). †P < 0.05, ‡P < 0.01, ¶P < 0.001 compared to EFS.

Figure 5. Effect of neostigmine on EFS-evoked gastric fundus smooth muscle contraction and CPI-17 and MYPT1 phosphorylation

A, gastric fundus smooth muscle tone is increased by neostigmine and is completely blocked by atropine. B, neostigmine augments the contraction evoked by EFS. C, the enhanced contractile response evoked by EFS in the presence of neostigmine is inhibited by SAR1x; representative traces. Upper panels, mean ratios of the pT38/CPI-17 density values (±SD) (D), the pT853/MYTP1 density values (±SD) (E), and the pT696/MYPT1 density values (±SD) (F) without or with EFS, or EFS in the presence of neostigmine (neo; D, E and F), atropine (D, E and F), or SAR1x (E and F); lower panels, representative Western blots. n= 5, duplicate blots. *P < 0.05, #P < 0.01, §P < 0.001 compared to control (white bars). †P < 0.05, ‡P < 0.01, ¶P < 0.001 compared to 10 Hz/5 s (black bars).

Figure 6. Effect of the absence of ICCs on EFS-evoked gastric fundus smooth muscle contraction and CPI-17 and MYPT1 phosphorylation

A, representative traces showing the EFS-evoked contractions of gastric fundus smooth muscles in the presence of l-NAME and MRS2500 from wild-type C57BL/6 mice and W/W^v mice, n= 5. Upper panels, mean ratios of the pT38/CPI-17 density values (±SD) (B), the pT853/MYTP1 density values (±SD) (C), and the pT696/MYPT1 density values (±SD) (D) in gastric fundus smooth muscles from wild-type C57BL/6 mice and W/W^v mice without or with EFS, or EFS in the presence of atropine; lower panels, representative Western blots. E, representative trace showing that neostigmine increases W/W^v gastric fundus smooth muscle tone, and augments the contraction evoked by EFS. F, upper panel, mean ratios of the pT38/CPI-17 density values (±SD) from W/W^v gastric fundus smooth muscles without or with EFS, neostigmine (neo), or EFS in the presence of neostigmine, or atropine. G, upper panel, mean ratios of the pT853/MYPT1 density values (±SD) from W/W^v gastric fundus smooth muscles without or with EFS, neostigmine (neo), or EFS in the presence of neostigmine, SAR1x, or atropine. n= 5, duplicate blots, *P < 0.05, #P < 0.01, §P < 0.001 compared to controls. †P < 0.05, ‡P < 0.01, ‡P < 0.01 compared to 10 Hz/5 s, ^fP < 0.01 compared to 10 Hz/5 s plus neostigmine, ¶P < 0.001 compared to 10 Hz/5 s plus neostigmine.

Figure 7. Effects of CCh treatment or EFS on LC20 and CaD phosphorylation in gastric fundus smooth muscles

A, upper panel, mean ratios of the pS19/LC20 density values (±SD) in the absence or presence of CCh or neostigmine (neo), or CCh in the presence of atropine, or SAR1x; lower panel, representative Western blot. B, upper panel, mean ratios of the pS19/LC20 density values (±SD) without or with EFS or neostigmine, or EFS in the presence of neostigmine, SAR1x, or atropine; lower panel, representative Western blot. C, upper panel, mean ratios of the pS789/CaD density values (±SD) without or with CCh or EFS; lower panel, representative Western blot. n= 4, duplicate blots. *P < 0.05 compared to control. †P < 0.05, ‡P < 0.01 compared to CCh. CaD, caldesmon.

Figure 8. Model of a neuroeffector junction in GI smooth muscles

A, stimulation of cholinergic neurons releases ACh into the synaptic-like space (∼20 nm) between the enteric motor neuron (EMN) and the interstitial cell of Cajal (ICC), where it binds primarily to M3 receptors on the ICC and activates signalling pathways that stimulate non-selective cation currents (NSCC) and Ca²⁺-activated Cl^- channels (CaCC) to depolarize (Dep) the smooth muscle cell (SMC) and activate voltage-dependent Ca²⁺ channels (VDCC). Ca²⁺ entry results in contraction and CPI-17 phosphorylation by PKC. Cholinesterases (AChE) also rapidly hydrolyse ACh, limiting the spread of ACh. B, bath-applied CCh ‘floods’ the entire tissue, directly activating M3 receptors on the SMC in addition to M3 receptors on the ICC. Along with VDCC-dependent Ca²⁺ entry and CPI-17 phosphorylation, CCh binding to SMC M3 receptors activates the G_q and RhoA Ca²⁺ sensitization pathways to phosphorylate MYPT1 and further stimulate contraction. TTx, tetrodotoxin; IP3, inositol trisphosphate.