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Sensitization of the contractile system of canine colonic smooth muscle by agonists and phorbol ester
K Sato1, R Leposavic, N G Publicover
1Department of Physiology, University of Nevada School of Medicine, Reno 89557-0046.
Abstract:
1. Sensitization of the contractile system in response to combinations of excitatory agonists acetylcholine (ACh), methacholine, histamine and neurokinin A (NKA) was investigated in colonic circular smooth muscle of dog, NKA (1 nM) potentiated the contractile response to 1 microM ACh, but did not increase the fura-2 fluorescence ratio (R340/380). Contraction in response to low concentrations of either methacholine or histamine was potentiated significantly by 0.1 microM 4-phorbol 12,13-dibutyrate (PDBu), suggesting that activation of protein kinase C can potentiate contraction at threshold concentrations of agonists. 2. Variability in the sensitivity of the contractile system to Ca2+ was demonstrated over a range of agonist concentrations. KCl, ACh, histamine and NKA each produced a concentration-dependent increase in the amplitude of phasic contractions and R340/380. However, ACh, histamine and NKA each induced maximal increases in R340/380 at concentrations less than that needed to induce maximum force. 3. In depolarized muscles, NKA (50 nM) and PDBu (1 microM) each increased the magnitude of tonic contraction with no change or a decrease in both R340/380 and myosin light chain phosphorylation. In alpha-toxin-permeabilized fibres, 0.1 microM PDBu and 1 microM NKA shifted the Ca(2+)-force response to the left. Ca(2+)-induced contractions were also potentiated by 100 microM GTP-gamma-S or 1 microM NKA plus 10 microM GTP. Potentiation of contraction by NKA and GTP was antagonized by 10 microM GDP-beta-S. 4. The results suggest that endogenous agonists acting via G-proteins sensitize the contractile element of colonic smooth muscle in part by activation of protein kinase C. In some cases, sensitization may be secondary to increased myosin phosphorylation (ACh), but in other cases it appears to be independent of increased myosin light chain phosphorylation (NKA and PDBu). Therefore regulatory mechanisms in addition to myosin phosphorylation contribute to the apparent sensitization of the contractile system to Ca2+.
Insights
Neurokinin A and protein kinase C activation sensitize colonic smooth muscle. This sensitization enhances contractile responses to agonists, partly through mechanisms beyond myosin phosphorylation, involving G-protein signaling.
Area of Science:
- Gastrointestinal Physiology
- Smooth Muscle Pharmacology
- Cellular Signaling
Background:
- Colonic smooth muscle contraction is regulated by various excitatory agonists.
- Agonist-induced sensitization of contractile responses is a key physiological process.
- The precise mechanisms underlying sensitization, particularly involving protein kinase C and G-proteins, require further elucidation.
Purpose of the Study:
- To investigate the sensitization of dog colonic circular smooth muscle to excitatory agonists.
- To determine the role of protein kinase C (PKC) and G-proteins in agonist-induced sensitization.
- To explore the relationship between sensitization, calcium sensitivity, and myosin light chain phosphorylation.
Main Methods:
- Studied contractile responses and fura-2 fluorescence ratios in isolated colonic smooth muscle.
- Utilized agonists like acetylcholine (ACh), methacholine, histamine, and neurokinin A (NKA).
- Employed 4-phorbol 12,13-dibutyrate (PDBu) to activate PKC and GTP analogs in permeabilized muscle fibers.
Main Results:
- NKA potentiated ACh-induced contractions without increasing intracellular calcium.
- PKC activation by PDBu potentiated contractions at low agonist concentrations.
- NKA and PDBu increased tonic contraction magnitude independently of changes in calcium or myosin phosphorylation; they also shifted the Ca2+-force relationship leftward in permeabilized fibers.
Conclusions:
- Endogenous agonists, via G-proteins, sensitize colonic smooth muscle contractile elements, partly through PKC activation.
- Sensitization mechanisms involve pathways additional to myosin light chain phosphorylation.
- These findings highlight complex regulatory mechanisms contributing to colonic smooth muscle contractility.