Pharmacological evidence for the contribution of polyamines as mediators of the transcriptional component involved in smooth muscle relaxation elicited by forskolin

Abstract

To study whether cAMP-dependent transcriptional effect and polyamines might play a modulatory role on smooth muscle, the effect of forskolin on KCl (60 mM)-induced contractions in isolated rat uterus and its modification by inhibitors of cAMP-dependent protein kinase (PKA) (Rp-cAMPS and TPCK), transcription (actinomycin D), protein synthesis (cycloheximide) and ornithine decarboxylase (α-difluoromethyl-ornithine, DFMO), and a polyamine (spermine) have been assayed. Forskolin (0.1 to 6 μM) induced concentration-dependent relaxation on KCl-induced tonic contractions in rat uterus (IC 50: 0.55 ± 0.12 μM) which was antagonized (p < 0.05) by Rp-cAMPS (30 μM), TPCK (3 μM), cycloheximide (300 μM), actinomycin D (4 and 12 μM) and TPCK (3 μM) plus actinomycin D (12 μM). The IC 50 values of forskolin in the presence of these drugs were 3.75 ± 1.53 μM, 12.08 ± 8.18 μM, 6.88 ± 5.02 μM, 3.80 ± 2.35 and 5.31 ± 2.80 μM, and 4.26 ± 3.65 μM respectively. Furthermore, DFMO (10 mM) also shifted the relaxation curve to forskolin to the right (IC 50: 3.06 ± 2.66 μM, p < 0.05) but DFMO (10 mM) plus actinomycin D (12 μM) (IC 50: 1.78 ± 1.33 μM) did not. However, DFMO (10 mM) and actinomycin D (12 μM) did not antagonize the spermine (1–30 mM)-elicited relaxation (IC 50s: 7.8 ± 0.7 mM vs 7.28 ± 1.4 mM and 4.67 ± 0.44 mM in the presence of DFMO and actinomycin D, respectively). Moreover, spermine (1 mM) did not decrease the forskolin induced relaxation and counteracted the antagonism produced by actinomycin D and DFMO. Our results suggest that, in rat uterus, forskolin: a) produced cAMP-dependent relaxation, as this is antagonized by Rp-cAMP and TPCK, and b) increased the activity of ornithine decarboxylase, as this is inhibited by DFMO. Therefore, polyamines could be the mediator of the cAMP-dependent transcriptional component involved in forskolin relaxation, since, as mentioned, DFMO antagonized this relaxation and spermine counteracted the displacement produced by DFMO and actinomycin D. Thus, a plasma membrane-nucleus interaction might, at least partially, explain the mechanisms involved in forskolin induced relaxation in smooth muscle of rat uterus under the present experimental conditions.