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Comparative Study
. 2005 Jun;145(3):353-63.
doi: 10.1038/sj.bjp.0706181.

The mechanism underlying the contractile effect of a chemotactic peptide, formyl-Met-Leu-Phe on the guinea-pig Taenia coli

Hiroshi Kawata  1 , Katsuya Hirano, Junji Nishimura, Chiharu Kubo, Hideo Kanaide
Affiliations
Comparative Study

The mechanism underlying the contractile effect of a chemotactic peptide, formyl-Met-Leu-Phe on the guinea-pig Taenia coli

Hiroshi Kawata et al. Br J Pharmacol. 2005 Jun.

Abstract

1 The contractile mechanism of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was investigated in the guinea-pig Taenia coli, by simultaneously monitoring the changes in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force. 2 fMLP induced a significant elevation of [Ca(2+)](i) and force at concentrations higher than 10 nM. The maximal response was obtained at a concentration of higher than 1 microM. 3 fMLP (10 microM) augmented the force development induced by a stepwise increment of the extracellular Ca(2+) concentration during 60 mM K(+) depolarization, while it had no effect on the [Ca(2+)](i) elevation, and thus produced a greater force for a given elevation of [Ca(2+)](i) than 60 mM K(+) depolarization. 4 The removal of extracellular Ca(2+) completely abolished the fMLP-induced contraction. The fMLP-induced [Ca(2+)](i) elevation was inhibited substantially but not completely by 10 microM diltiazem, partly by 10 microM SK&F 96365, and completely by their combination. 5 Y27632, a specific inhibitor of rho-kinase, had no significant effect on the fMLP-induced [Ca(2+)](i) elevation and force development. 6 Chenodeoxycholic acid, a formyl peptide receptor antagonist, specifically abolished the fMLP-induced contraction but not high K(+)- or carbachol-induced contractions. 7 A dual lipoxygenase/cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, a nonselective leukotriene receptor antagonist, and a selective type 1 cysteinyl-containing leukotriene receptor antagonist specifically reduced the fMLP-induced contraction. 8 We suggest that the low-affinity-type fMLP receptor and lipoxygenase metabolites of arachidonic acid are involved in the fMLP-induced contraction in the guinea-pig T. coli. This contraction mainly depends on the [Ca(2+)](i) elevation due to Ca(2+) influx and the enhancement of Ca(2+) sensitivity in the contractile apparatus.

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Figures

Figure 1
Figure 1
Changes in the [Ca2+]i and force induced by fMLP in the strips of guinea-pig T. coli. (a) A representative recording showing the changes in [Ca2+]i and force induced by 10 μM fMLP in normal PSS. The level of [Ca2+]i and force at rest (5.9 mM K+–PSS) and at the steady state of contraction induced by 60 mM K+ depolarization were assigned to be 0 and 100%, respectively. (b) Concentration–response curves for the fMLP-induced maximal elevation of [Ca2+]i and force in the guinea-pig T. coli. The data are the mean±s.e.m. (n=5–9). *Significant (P<0.05).
Figure 2
Figure 2
Effect of fMLP on the contraction induced by the cumulative application of the extracellular Ca2+ during 60 mM K+ depolarization in the guinea-pig T. coli. (a) A representative recording showing the changes in [Ca2+]i and force induced by the stepwise increment of [Ca2+]o during the 60 mM K+ depolarization in the absence of fMLP. The numbers noted by the arrow heads indicate [Ca2+]o obtained at each step. The level of [Ca2+]i and force at rest (5.9 mM K+–PSS) and at the steady state of contraction induced by the first stimulation with 60 mM K+ depolarization was assigned to be 0 and 100%, respectively, 0.3 G, 0.3 mM EGTA. (b) Summary of the changes in [Ca2+]i (left) and force (right) induced by the cumulative application of [Ca2+]o during 60 mM K+ depolarization in the presence and absence of 10 μM fMLP. fMLP was applied 5 min prior to and during the contraction induced by the application of extracellular Ca2+. The data are the mean±s.e.m. (n=4–5). *Significantly (P<0.05); NS, not significantly (P>0.05) different from the values obtained without fMLP. (c) The [Ca2+]i–force relation curves reconstructed from the data in (b).
Figure 3
Figure 3
Contractions induced by fMLP and carbachol in the absence of the extracellular Ca2+. (a–c) Representative recordings showing the changes in [Ca2+]i and force induced by 10 μM fMLP (a) and 10 μM carbachol (b and c) in the Ca2+-free PSS containing 0.3 mM EGTA without (b) and with (c) 10 μM fMLP in the guinea-pig T. coli. After recording a reference response to 60 mM K+, the strips were exposed to the Ca2+-free PSS for 3 min, and then were stimulated by fMLP and carbachol. (d) A summary of the increases in [Ca2+]i and force induced by 10 μM carbachol in the Ca2+-free PSS containing 0.3 mM EGTA with and without 10 μM fMLP. The [Ca2+]i and force were expressed as a percentage, assigning the values in normal (5.9 mM K+) PSS and 60 mM K+ PSS to be 0 and 100%, respectively. The bottom and top of each column represent the levels of [Ca2+]i and force obtained just before and at the maximal elevation after stimulation with carbachol, respectively. The data are the mean±s.e.m. (n=5–6). NS, not significantly different (P>0.05).
Figure 4
Figure 4
Effects of diltiazem, SK&F 96365 and Y27632 on the contractions induced by fMLP and carbachol in the guinea-pig T. coli. Representative recordings showing the changes in [Ca2+]i and force induced by 10 μM fMLP (a–d) and 10 μM carbachol (e–h) in the normal PSS without (a and e) and with 10 μM diltiazem (b and f), 10 μM SK&F 96365 (c and g), and 10 μM Y27632 (d and h). Diltiazem, SK&F 96365 and Y27632 were applied 10 min before and during the contraction induced by fMLP and carbachol. The [Ca2+]i and force were expressed as a percentage, assigning the values in normal PSS and 60 mM K+ PSS to be 0 and 100%, respectively.
Figure 5
Figure 5
Summary of the effects of diltiazem, SK&F 96365, and Y27632 on the increases in [Ca2+]i and force induced by fMLP and carbachol. The effect of 10 μM diltiazem, 10 μM SK&F 96365, 10 μM diltiazem plus 10 μM SK&F 96365, and 10 μM Y27632 on the fMLP-induced contraction were evaluated at 15 min after the initiation of the contraction. The levels of [Ca2+]i and force obtained at rest (in 5.9 mM K+, 2.5 mM Ca2+-containing normal PSS) and those obtained at the steady state of the 60 mM K+-induced contraction were assigned values of 0 and 100%, respectively. The level of the bottom and top of each column indicate the level of [Ca2+]i and force obtained just before and 15 min after initiating the contraction. The data are the mean±s.e.m. of the number of experiments as indicated in parentheses. *Significantly (P<0.05); NS, not significantly (P>0.05) different from the control values.
Figure 6
Figure 6
Concentration-dependent effects of chenodeoxycholic acid on the changes in [Ca2+]i and force induced by fMLP, carbachol, and 60 mM K+ depolarization in the guinea-pig T. coli. After recording a reference response to 60 mM K+ depolarization, the strips were pretreated with the indicated concentrations of chenodeoxycholic acid for 10 min, and then stimulated with 10 μM fMLP, 10 μM carbachol, and 60 mM K+ depolarization in the presence of chenodeoxycholic acid. The levels of [Ca2+]i and force obtained at the peak of the contraction are shown. *Significantly (P<0.05); NS, not significantly (P>0.05) different from the control value obtained in the absence of chenodeoxycholic acid. The data are the means±s.e.m. (n=3–5). The [Ca2+]i and force were expressed as a percentage, assigning the values in normal PSS and 60 mM K+ PSS to be 0 and 100%, respectively.
Figure 7
Figure 7
Involvement of lipoxygenase metabolites in the fMLP-induced contraction in the guinea-pig T. coli. The effect of 30 μM phenidone, 1 μM nordihydroguaiaretic acid, 100 μM aspirin, 10 μM indomethacin, 1 μM ONO-1078, 0.3 μM MK-571, 10 μM BAY u9773, and 1 μM U-75302 on the fMLP-induced contraction were evaluated at the maximal elevation of [Ca2+]i and force. Note that the effect of indomethacin on [Ca2+]i was not evaluated because indomethacin caused an artifact in fluorescence studies. The levels of [Ca2+]i and force obtained at rest (in 5.9 mM K+, 2.5 mM Ca2+-containing normal PSS) and those obtained at the steady state of the 60 mM K+-induced contraction were assigned values of 0 and 100%, respectively. The data are the mean±s.e.m. of the number of experiment as indicated in parentheses. *Significantly (P<0.05); NS, not significantly (P>0.05) different from the control values.
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