Effect of spermine and alpha-difluoromethylornithine on KCl- and CaCl2-induced contraction in rat uterine smooth muscle.

Abstract

1. The effect of a polyamine, spermine (0.3-30 mM), and an ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO; 1-10 mM), given alone and in combination were studied on KCl (33, 60 or 90 mM)-induced tonic contraction and on the cumulative concentration-response curves elicited by CaCl2 (30 microM to 10 mM) in a depolarizing (with 33, 60 or 90 mM of KCl) calcium-free medium in isolated rat uterus. 2. Spermine elicited a concentration-dependent relaxation on KCl 33 mM (IC50 = 2.18 +/- 0.37 mM, n = 6), 60 mM (IC50 = 7.80 +/- 0.79 mM, n = 11) and 90 mM KCl (IC50 = 29.55 +/- 4.08 mM, n = 7) induced tonic contraction. The IC50 values were significantly different (P < 0.01). 3. DFMO relaxed the tonic contraction induced by 33 mM of KCl (Emax = 80.70 +/- 13.01%) but only relaxed the contractions induced by 60 and 90 mM of KCl to 23.60 +/- 4.60% and 16.90 +/- 4.10%, respectively. DFMO (10 mM) did not modify the concentration-dependent relaxation elicited by spermine on KCl (60 mM)-induced tonic contraction. 4. KCl (33, 60 or 90 mM) did not produce contraction in a calcium-free medium, but enabled CaCl2 (30 microM to 10 mM) to induce cumulative concentration-dependent contraction of the rat uterus. The EC50 values for CaCl2 were: 0.74 +/- 0.08 (n = 12), 0.34 +/- 0.03 (n = 14) and 0.48 +/- 0.02 (n = 12) mM in medium with 33, 60 or 90 mM of KCl, respectively. 5. Spermine (1 mM) and DFMO (1 mM) did not modify the concentration-response curves induced by CaCl2 in medium with 33 mM of KCl. Higher concentrations of spermine (3 mM) and DFMO (10 mM) strongly reduced the contractile effect of CaCl2 to 35.69 +/- 3.96% (n = 6) and 40.14 +/- 10.74% (n = 6). This inhibitory effect of spermine and DFMO was prevented by increasing KCl concentration in the medium to 60 or 90 mM. Thus, the Emax of CaCl2 in the presence of spermine (3 mM) was 66.26 +/- 6.96% and 89.02 +/- 2.89% in a 60 and 90 mM KCl medium, respectively. In the presence of DFMO (10 mM) the Emax of CaCl2 reached 100% when KCl was increased. 6. Spermine (1 mM) plus DFMO (1 mM) and spermine (1 mM) plus DFMO (10 mM) produced a synergic inhibitory effect of CaCl2-induced contraction in medium with 33 mM of KCl. Spermine (3 mM) plus DFMO (10 mM) produced a total inhibition of CaCl2-induced contraction. 7. The inhibitory effect of spermine (1 mM) plus DFMO (10 mM) was also prevented by increasing KCl concentration in the medium to 60 or 90 mM. The Emax for CaCl2 (10.9 +/- 5.5%) in the presence of KCl 33 mM increased up to 93.2 +/- 2.1% and 96.3 +/- 1.2% when the KCl concentration in the medium was enhanced to 60 or 90 mM. 8. Our results suggest that a calcium inhibitory effect of spermine and DFMO in isolated rat uterus could be produced, since this was prevented by depolarization, as a result of the increase of KCl concentration in the medium.