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Receptor-dependent G protein-mediated Ca2+ sensitization in canine airway smooth muscle
K Iizuka1, K Dobashi, A Yoshii
1First Department of Internal Medicine, Gunma University Faculty of Medicine, School of Medicine, Japan. Iizukak@sb.gunma-u.ac.jp
Abstract:
To determine the mechanisms of receptor-dependent Ca2+ sensitization in airway smooth muscle, canine tracheal smooth muscle (CTSM) was permeabilized with alpha-toxin or beta-escin. Although the effects of 5-hydroxytryptamine (100 microM), histamine (100 microM), and the thromboxane A2 analogue U-46619 (100 microM) were negligible, carbachol (100 microM) and endothelin-1 (ET-1, 1 microM) evoked additional contractions of 47.0 +/- 5.90% and 25.0 +/- 5.37% (n = 6) at pCa 6.7 with GTP (3 microM) (normalized to the maximum contraction at pCa 4.5) in alpha-toxin-permeabilized CTSM. GDP-beta-S (1 mM) reversed the carbachol and ET-1 responses completely. GTP-gamma-S (30 microM) and 4 beta-phorbol 12,13-dibutyrate (PDBu, 3 microM) increased the Ca2+ sensitivity (median effective pCa) of contraction by 1.8- and 4.4-fold, respectively (n = 4-11, P < 0.05). The effects of saturating concentrations of GTP-gamma-S and PDBu were additive. A synthetic peptide (T2) corresponding to the actin-binding site of calponin caused a dose-dependent contraction of beta-escin permeabilized CTSM, with the peak effect (25 +/- 4%, n = 4) at 1200 microM, PDBu (3 microM) caused contraction of the T2 peptide-treated CTSM. In conclusion, Ca2+ sensitization of CTSM depends on receptor type and is mediated by G proteins and protein kinase C whose effects are additive, with a partial contribution by calponin.
Insights
Receptor-dependent calcium sensitization in airway smooth muscle involves G proteins and protein kinase C, with a partial role for calponin. These pathways contribute to muscle contraction, with varying effects based on receptor type.
Area of Science:
- Physiology
- Pharmacology
- Cell Biology
Background:
- Airway smooth muscle (ASM) contraction is regulated by calcium (Ca2+).
- Receptor-dependent Ca2+ sensitization mechanisms in ASM are not fully understood.
- Investigating these mechanisms is crucial for understanding airway hyperresponsiveness.
Purpose of the Study:
- To elucidate the mechanisms of receptor-dependent Ca2+ sensitization in canine tracheal smooth muscle (CTSM).
- To identify the roles of G proteins and protein kinase C (PKC) in this process.
Main Methods:
- CTSM cells were permeabilized using alpha-toxin or beta-escin.
- Muscle contractions were measured in response to various agonists (carbachol, endothelin-1, U-46619) and signaling modulators (GTP, GDP-beta-S, GTP-gamma-S, PDBu).
- The effect of a calponin-derived peptide (T2) on contraction was also assessed.
Main Results:
- Carbachol and endothelin-1 evoked contractions in alpha-toxin-permeabilized CTSM, reversed by GDP-beta-S.
- GTP-gamma-S and PDBu significantly increased Ca2+ sensitivity of contraction in a dose-dependent manner.
- The effects of GTP-gamma-S and PDBu were additive, and PDBu induced contraction in T2 peptide-treated muscle.
Conclusions:
- Ca2+ sensitization of CTSM is receptor-dependent.
- G proteins and PKC mediate Ca2+ sensitization, with additive effects.
- Calponin contributes partially to this sensitization process.