Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl 2

Abstract

1. 1. The effects of the flavonoids genistein (3–60 μM), kaempferol (3–60 μM) and quercetin (1–100 μM) on KCI (60 μM)-induced tonic contraction in rat uterus and their modifications with the inhibitor of cAMP-dependent protein kinases (TPCK, 3 μM), the inhibitor of ornithine decarboxylase [α-difluoromethyl ornithine (DFMO), 10 mM] and the polyamine spermine (1 mM) have been assayed. The effects of the three flavonoids were also studied on the contraction elicited by CaCl 2 (30 μM to 10 mM) on rat uterus incubated in medium lacking calcium and supplemented with 33, 60 or 90 μM of KCI. For comparison, the effects of the calcium channel blockers nifedipine and verapamil and the activator of adenylyl cyclase forskolin were assayed on contractions induced by KCl and CaCl 2. 2. 2. Genistein (IC 50: 20.2± 1.0 μM, n=11), kaempferol (IC 50:10.1±0.8 μM, n=8) and quercetin (IC 50:13.2±0.5 μM, n=8) relaxed the tonic contraction induced by KCI (60 mM) in a concentration-dependent way. Verapamil (IC 50:70.1±5.8 μM, n=7), nifedipine (IC 50:8.4±0.7 nM, n=6) and forskolin (IC 50: 0.62±0.08 μM, n=14) also relaxed the KCl-induced contraction. TPCK (3 μM) significantly antagonized the effect of quercetin, kaempferol and forskolin ( P<0.01) but did not modify the effect of genistein. 3. 3. Spermine (1 mM) increased the effects of genistein and verapamil and antagonized the effect of quercetin but did not modify those of kaempferol and forskolin. DFMO (10 mM) did not modify the effect of quercetin but increased that of genistein and antagonized those of kaempferol and forskolin. The addition of spermine (1 mM) plus DFMO (10 mM) antagonized the effect of quercetin. Spermine counteracted the effect of DFMO on forskolin but not on genistein. 4. 4. KCI (33, 60 or 90 mM) did not produce contraction in calcium-free solution, but CaCl 2 (30 μM to 10 mM) induced concentration-dependent contraction after depolarizing with KC1. The EC 50 values for CaCl 2 were: 0.74±0.08 ( n=12), 0.34±0.03 ( n=14) and 0.48±0.02 ( n=12) mM in a medium with 33, 60 or 90 mM of KCI, respectively. 5. 5. Genistein (20 μM), kaempferol (10 μM), quercetin (15 μM), verapamil (70 nM), nifedipine (10 nM) and forskolin (0.5 μM) inhibited the concentration-response curve to CaCl 2 in medium supplemented with 33, 60 or 90 mM of KCI. The effect of kaempferol was independent of the concentration of KCl in the incubation medium. However, the inhibitory effect of genistein on CaCl 2-induced contraction was inversely related to the concentration of KCI in the medium. On the contrary, the effect of quercetin was directly related to the concentration of KCl in the medium. 6. 6. The antagonism of verapamil, nifedipine and forskolin on CaC1 2-induced contraction seems to be related to the degree of depolarization because increasing the KCl in the medium counteracted their effects. 7. 7. Our results suggest that (1) cAMP contributes to the relaxant effects of quercetin and kaempferol on KCI (60 mM)-induced tonic contraction; (2) polyamines are involved in the relaxant effects of forskolin and kaempferol on KCl-induced tonic contraction but not on CaCl 2-induced contraction in the depolarized uterus, and (3) the flavonoids assayed also possess a calcium antagonist action but show a different behavior toward the calcium channel blockers and the cAMP enhancer forskolin.