Calmodulin inhibitors induce spinal analgesia in rats.

Calcium is an important intracellular messenger that interacts with Ca(2+)-binding proteins, such as calmodulin (CaM), to activate several intracellular enzymes. The involvement of Ca2+ in the transmission of nociceptive signals has been demonstrated at the spina level. Specifically, spinal sensitization induced by persistent nociceptive stimulation seems to be related to an increase of cytosolic calcium and the subsequent activation of several enzymes, some of which are Ca2+/CaM dependent. In order to elucidate the possible implication of calmodulin in these pain processes, we have studied the effect of two calmodulin inhibitors (W-7 and calmidazolium) or the formalin and tail-flick tests in rats after their intrathecal administration. Antinociceptive effects were observed in both tests by injecting 0.12-1 mumol/rat of calmidazolium and 0.25-2 mumol/rat of W-7. Calmidazolium was more potent than W-7 in inhibiting both phases of the formalin test, whereas lower doses of W-7 in comparison to calmidazolium affected the tail-flick latencies. In addition, both drugs induced, at high doses, a muscular flaccidity of the hindlimbs that impaired normal walking in the rats. This effect caused; significant reduction of the rotarod performance when 1 mumol/rat of calmidazolium or 2 mumol/rat of W-7 were injected. Overall, our results show that calmodulin inhibitors are capable of producing spinal analgesia on phasic and tonic noxious stimuli in rats, thus rendering them a promising potential as analgesics.

Increases in ornithine decarboxylase activity in the positive inotropism induced by androgens in isolated left atrium of the rat.

It is well established that the intracellular receptors of androgens act as transcription factors upon their activation by androgen binding. However, a growing number of studies have associated androgens with rapid biological responses independent of their classical action mechanism. In this sense, 5alpha- and 5beta-dihydrotestosterone elicited a rapid positive inotropism in the isolated left atrium of the rat via cAMP-dependent mechanisms that may involve genomic effects. In addition, polyamines are mediators of several biological actions including those acute and long-term effects induced by androgens in the heart. The present study analyzed the role of polyamine synthesis in the cardiotonic effect of androgens in the left atrium of male Wistar rats, electrically stimulated (0.5 Hz, 5 ms and supramaximal voltage) and placed in an organ bath in 10 ml of Tyrode's solution. Incubation in the organ bath with an inhibitor of ornithine decarboxylase activity, alpha-difluoromethylornithine 10 mM, significantly decreased the positive inotropism induced by 5alpha- and 5beta-dihydrotestosterone (0.1-100 microM). This suggests that ornithine decarboxylase seems to be involved in androgen-induced positive inotropism. Furthermore, 6-min exposure to 5alpha- or 5beta-dihydrotestosterone significantly increased the activity of ornithine decarboxylase from 61.81+/-7.53 (control) to 93.28+/-9.45 and 80.28+/-12 pmol/h/mg of protein, respectively. Northern blot analysis showed that 5alpha- and 5beta-dihydrotestosterone did not modify the level of expression of the ornithine decarboxylase gene. Therefore, our results suggest that polyamine synthesis might be involved in the positive inotropism elicited by androgens through the stimulation of ornithine decarboxylase activity without changes in the expression of the ornithine decarboxylase gene.

Mechanism of mifepristone-induced spasmolytic effect on isolated rat uterus.

Mifepristone, a synthetic 19-norsteroid, relaxed the KCl-induced tonic contraction in isolated rat uterus in a concentration-dependent way and CaCl2 (0.1 to 10 mM) counteracted it. This effect was similar to other steroids although the mechanisms involved are unclear. Before adding the contracturant, tissue was incubated with actinomycin D (10 microM), cycloheximide (300 microM), TPCK (3 and 10 microM), Rp-cAMPS (30 microM), DDA (100 microM) and H-7 (1 microM). None of these modified the relaxing effect of mifepristone. Incubation with drugs that interfere with cGMP such as a nucleotide analogue DDG (100 microM), a soluble guanylyl cyclase inhibitor ODQ (1 microM) and an inhibitor of protein kinase G 8pCPTcGMPS (1 microM) significantly modified the effect of mifepristone, increasing its IC50.

Genomic and non-genomic effects of steroidal drugs on smooth muscle contraction in vitro.

The effect of steroidal drugs on KCl (60 mM)-induced tonic contraction in in vitro rat uterus has been assayed. Ouabain had no effect and aldosterone only relaxed the KCl contraction up to 27 +/- 7.3%. However, estradiol, testosterone, progesterone, cortisol and alphaxalone relaxed the contraction in a dose-dependent way, and CaCl2 (0.1 to 6 mM) counteracted this effect. Cycloheximide (1 and 10 micrograms/ml) did not modify the effect of progesterone, testosterone or alphaxalone. Cycloheximide, but not actinomycin D (5 micrograms/ml), reduced the effect of cortisol. Both cycloheximide and actinomycin D shifted righward the relaxing effect of estradiol. This suggests that the steroidal relaxing effect in smooth muscle is preferably non-genomic and at plasma membrane level. However, cortisol and estradiol also act at intracellular levels and induce transcriptional (estradiol) or non-transcriptional (cortisol) effects.

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

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 (alpha-difluoromethyl-ornithine, DFMO), and a polyamine (spermine) have been assayed. Forskolin (0.1 to 6 microM) induced concentration-dependent relaxation on KCl-induced tonic contractions in rat uterus (IC50: 0.55 +/- 0.12 microM) which was antagonized (p<0.05) by Rp-cAMPS (30 microM), TPCK (3 microM), cycloheximide (300 microM), actinomycin D (4 and 12 microM) and TPCK (3 microM) plus actinomycin D (12 microM). The IC50 values of forskolin in the presence of these drugs were 3.75 +/- 1.53 microM, 12.08 +/- 8.18 microM, 6.88 +/- 5.02 microM, 3.80 +/- 2.35 and 5.31 +/- 2.80 microM, and 4.26 +/- 3.65 microM respectively. Furthermore, DFMO (10 mM) also shifted the relaxation curve to forskolin to the right (IC50: 3.06 +/- 2.66 microM, p<0.05) but DFMO (10 mM) plus actinomycin D (12 microM) (IC50: 1.78 +/- 1.33 microM) did not. However, DFMO (10 mM) and actinomycin D (12 microM) did not antagonize the spermine (1-30 mM)-elicited relaxation (IC50s: 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.

Mechanisms involved in kaempferol-induced relaxation in rat uterine smooth muscle.

The effect of kaempferol on KCI (60 mM)-induced tonic contraction in isolated rat uterus and its modification by inhibitors of cAMP-dependent protein kinase (PKA) (Rp-cAMPS and TPCK), phosphodiesterase (papaverine), adenylyl cyclase (2',3'-dideoxyadenosine, DDA), transcription (actinomycin D), protein synthesis (cycloheximide) and ornithine decarboxylase (alpha-difluoromethyl-ornithine, DFMO), as well as a polyamine, spermine, have been assayed. Kaempferol (3 to 60 microM) induced concentration-dependent relaxation on KCl-induced tonic contraction (IC50: 10.1 +/- 1.89 microM). This relaxing effect was antagonized (p<0.05) by Rp-cAMPS (10 microM), TPCK (3 microM), DDA (100 microM), actinomycin D (4 and 12 microM), cycloheximide (100 microM), DFMO (10 mM), actinomycin D (12 microM) plus TPCK and actinomycin D (12 microM) plus spermine (1 mM). Furthermore, the displacement obtained with actinomycin D plus DFMO was not statistically significant. Our results suggest that kaempferol through cAMP produces transcriptional events and polyamines are, at least partially, involved in the relaxant effect of kaempferol.

Effects of androgens on isolated rat uterus.

The 5 alpha- and 5 beta-dihydrotestosterone (DHT) androgens relax KCl-induced tonic contraction in rat uterus, in a dose-dependent way. The 5 alpha and 5 beta-DHT relaxing effect is counteracted by CaCl2 (0.1-10 mM). The 5 alpha-DHT, but not the 5 beta-DHT, effect was reduced by cycloheximide (10 micrograms/ml) and by actinomycin D (5 micrograms/ml). Flutamide at 10(-6) M shifted the effect of 5 alpha-DHT to the right. However, other doses of flutamide or cyproterone acetate did not modify the effect of both androgens. We suggest a non-genomic effect of 5 alpha-DHT and 5 beta-DHT in rat uterus contraction but that intracellular and genomic actions play a part in the relaxing effect of 5 alpha-DHT.

Positive inotropism induced by androgens in isolated left atrium of rat: evidence for a cAMP-dependent transcriptional mechanism.

Steroid hormones exert their biological actions via intracellular receptors modulation of transcription. In addition, a number of molecular interactions, and the existence of membrane receptors in several tissues, support the hypothesis of nongenomic action of steroids. The androgens, 5alpha- and 5beta-dihydrotestosterone (0.1 to 100 microM), induce a rapid positive inotropism in the isolated left atrium of male Wistar rats whose time course of response might suggest that it is a non-genomic effect. However, the fact that the facilitation of contractility was inhibited by actinomycin D (5 microg/ml) and cycloheximide (10 microg/ml) indicates that a transcriptional component might play a role. The existence of a rapid functional genomic role would be somewhat surprising. However, rapid transcriptional mechanisms were also observed in certain cAMP-dependent responses. In the left atrium of rat, Rp-cAMPS (10 microM), a cAMP-dependent protein kinase inhibitor, antagonized 5alpha- but not 5beta-dihydrotestosterone-induced positive inotropism. The inhibition by Rp-cAMPS of isoproterenol- and forskolin-induced positive inotropism, and the fact that these cAMP-dependent effects were also inhibited by actinomycin D and cycloheximide, suggest that a cAMP-dependent transcriptional component may be partly involved in the positive inotropism induced by 5alpha-dihydrotestosterone. In addition, 5alpha-dihydrotestosterone might increase the basal adenylyl cyclase activity by acting on unoccupied beta-adrenoceptor-G-protein-adenylyl cyclase complexes, since the elicited inotropism was inhibited by a beta-blocker, atenolol (1 microM), a G-protein inhibitor, pertussis toxin (2 microg/ml, 3 h), and an adenylyl cyclase inhibitor, dideoxy-adenosine (10 microM).

Effect of Rp diastereoisomer of adenosine 3',5' cyclic-monophosphothioate on the cAMP-dependent relaxation of smooth muscle.

The effect of Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) on relaxation elicited by histamine (1-100 microM), forskolin (1-60 microM), papaverine (1-100 microM), vinpocetine (1-100 microM), rolipram (0.1-1 mM), Sp-cAMPS (10-300 microM), 8-BrcAMP (10 microM - 1 mM) and 8-BrcGMP (3 microM - 1 mM) of the previous vanadate-induced contraction was assayed. The effect of Rp-cAMPS on the relaxing effect produced by forskolin, papaverine, vinpocetine, rolipram, Sp-cAMPS and 8-BrcAMP in KCl-induced tonic contraction was also assayed. Histamine, forskolin, papaverine, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP, but not vinpocetine, relaxed the vanadate-induced contractions in rat uterus incubated in medium lacking calcium plus EDTA in a concentration-dependent way. Rp-cAMPS (1-300 microM) had no effect on vanadate contraction. However, it antagonized the relaxation elicited by histamine and papaverine, but not that of forskolin, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP. Forskolin, papaverine, vinpocetine, rolipram and 8-BrcAMP, but not Sp-cAMPS, relaxed the KCl-induced contraction. Rp-cAMPS antagonized the relaxation elicited by forskolin, papaverine and vinpocetine, but not that of rolipram and 8-BrcAMP. Our results suggest that: a) Rp-cAMPS is an effective PKA inhibitor that could be used to study the involvement of cAMP on drug-induced response in smooth muscle, and b) the effects of Sp-cAMPS, 8-BrcAMP and rolipram were independent of the activation of protein kinases.

Spasmolytic and calmodulin inhibitory effect of non-steroidal anti-inflammatory drugs in vitro.

The effect of several anti-inflammatory drugs (NSAIDs), the calmodulin inhibitor W-7 and cortisol on vanadate-induced tonic contraction and on calmodulin dependent cAMP-phosphodiesterase activity have been assayed. Indomethacin, diclofenac, phenylbutazone, mefenamic acid, naproxen, tolmetin, piroxicam, aspirin and W-7, but not metimazol, produce dose-dependent relaxation of vanadate-induced tonic contraction on isolated rat uterus. Cortisol relaxes the vanadate contraction up to 45%. None of the drugs assayed inhibit the basal activity of phosphodiesterase with concentrations lower than 1 mM. However, indomethacin, diclofenac, phenylbutazone, mefenamic acid, naproxen, piroxicam, aspirin and W-7 inhibit, in a concentration-dependent way, the calmodulin-stimulated activity of phosphodiesterase. The maximum inhibition achieved with tolmetin (1 mM) and cortisol (1 mM) was 38% and 24%, respectively. Metamizol has no effect on basal or/and stimulated phosphodiesterase. This, as far as we know, is the first description of relationship between NSAIDs and calmodulin-dependent processes and our results suggest that the inhibition of calmodulin with NSAIDs may be directly related to their pKa and liposolubility.

Extracellular and intracellular effects of polyamines on smooth muscle contractions.

The natural polyamines spermine, spermidine and putrescine, and their metabolic products N1-acetylspermine (N-AS) and 5'-deoxi-5'-methyl-thioadenosine (DMT), but not N1-acetylspermidine nor N-acetylputrescine, relax the KCl-induced contraction in rat uterus in a dose-dependent way. This relaxing effect is counteracted by CaCl2 (0.1-6 mM) but not by Bay K 8644. Spermine, N-AS and DMT also inhibit the contraction induced by methacholine and PGF2 alpha. Spermine, DMT and N-AS relax the vanadate-induced contraction in uterus incubated in calcium-free solution plus EDTA. However, in the vanadate contraction, the EC50s are higher than those obtained with other contracturants. These results suggest that polyamines inhibit smooth muscle contraction by action at plasma membrane level decreasing the influx of calcium. However, intracellular actions of polyamines could also be involved in their effects.

Effects of some inhibitors of arachidonic acid metabolism in rat uterus in vitro.

The effect of four eicosanoid synthesis inhibitors (ESIs): mepacrine (6 x 10(-6) to 10(-4) M), nordihydroguaiaretic acid (NDHGA, 10(-6) to 10(-5) M), indomethacin (2 x 10(-6) and 2 x 10(-5) M) and imidazole (10(-5) and 10(-4) M), were studied on contractions induced by cumulative doses (4 and 20 mU/ml) of oxytocin (OT) on the uterus of rats both in natural estrus and ovariectomized. ESIs were also assayed on contractions induced by carbachol (10(-4) M) and KCl (60 mM) in rat uterus under natural estrus, and OT (10 mU/ml)-induced contractions in rat uterus incubated in calcium-free EDTA treated medium. Mepacrine, NDHGA and indomethacin, but not imidazol, inhibited in a dose-dependent way contractions induced by OT in the uterus. The effect was higher in ovariectomized than in natural estrus rats. Mepacrine and NDHGA, but not indomethacin or imidazole, inhibited contractions induced by carbachol and relaxed tonic contractions to KCl (60 mM). Mepacrine, NDHG and indomethacin also inhibited tonic contractions by OT in calcium-free EDTA treated medium. Our results suggest that mepacrine, NDHGA and indomethacin, independently of inhibition of eicosanoids synthesis, reduce the entry of extracellular calcium and/or the release of intracellular calcium.

Effects of nonsteroidal antiestrogens in the in vitro rat uterus.

We have studied the effect of nonsteroidal antiestrogens on rat uterine contractions induced by oxytocin (8 nmol/l), methacholine (10 mumol/l), prostaglandin F2 alpha (1 mumol/l), KCl (60 mmol/l) and CaCl2 (6 mmol/l). In a concentration-dependent way, the antiestrogens tamoxifen, clomiphene, nafoxidine and ethamoxytriphetol inhibited the amplitude and frequency of the oxytocin-induced contractions and the contraction produced by CaCl2. At a concentration of 30 mumol/l the four drugs inhibited the contractions induced by methacholine and prostaglandin F2 alpha. They also relaxed the tonic contraction to KCl in a concentration-dependent way. This action was partially counteracted by CaCl2 (0.1-10 mmol/l). Bay k 8644 (0.3 nmol/l to 3 mumol/l) only partially reversed the inhibition by ethamoxytriphetol (0.1 mmol/l) of CaCl2 (6 mmol/l)-induced contractions. The steroidal antiestrogen, ICI 164,384, which lacks agonist activity, had an inhibitory effect (44 +/- 4%, n = 7) on KCl-induced contractions only at a concentration of 0.1 mmol/l. However, the quaternary analogue of tamoxifen (tamoxifen ethyl bromide) produced 86 +/- 3% relaxation of the KCl-induced contracture (IC50 1.52 +/- 0.1 mumol/l, n = 10) and this effect was counteracted by addition of CaCl2. Taken together the results indicate that the inhibitory effects of nonsteroidal antiestrogens on rat uterine contractions could be mediated by an action to block Ca2+ entry through an agonist action on extracellular estrogen receptors.

Estrogen and antiestrogen non-genomic effect in rat uterus contraction in calcium-free solution.

1. The effects of estrogens estradiol (E2, 10(-6)-10(-4) M) and diethylstilbestrol (DES, 10(-6)-10(-4) M) and the antiestrogens nafoxidine (N, 10(-6)-10(-4) M), tamoxifen (T, 10(-6)-6 x 10(-4) M), tamoxifen ethyl bromide (TEB, 10(-4) M) and ICI 164,384 (ICI, 10(-5) M) on tonic contractions induced by oxytocin (2 x 10(-8) M) or vanadate (3 x 10(-4) M) in rat uterus incubated in calcium-free EDTA treated solution have been assayed. 2. E2 and DES relaxed the tonic contraction induced by oxytocin in a dose dependent way (EC50: 1.11 +/- 0.01 x 10(-4) M and 1.5 +/- 0.07 x 10(-5) M). The vanadate-induced contraction only was relaxed with DES (57.62 +/- 2.38% at 10(-3) M). 3. The effect of DES on oxytocin contraction was unmodified by the protein synthesis inhibitor cycloheximide (10 micrograms/ml) and by the cyclooxygenase inhibitor indomethacin (3 x 10(-6) M), but enhanced by the intracellular calcium release inhibitor TMB-8 (10(-5) M). The antiestrogen tamoxifen (3 x 10(-5) M) promotes the relaxing effect of DES. 4. The antiestrogens N, and T, but not ICI, relaxed the oxytocin-induced contraction (EC50: 4.51 +/- 0.43 x 10(-5) M and 2.27 +/- 0.05 x 10(-4) M). TEB (10(-4) M) produces a relaxation of 74.5 +/- 2.11%. The vanadate contraction is also relaxed by T (EC50: 6.03 +/- 0.04 x 10(-4) M). 5. The effect of T on oxytocin contraction was unmodified with cycloheximide or TMB-8 but decreased with indomethacin.

Spasmolytic activity of a lipidic extract from Sabal serrulata fruits: further study of the mechanisms underlying this activity.

The mechanisms involved in the spasmolytic effect of a lipidic extract from Sabal serrulata fruits were investigated. The extract relaxed vanadate-induced contractions on rat uterus incubated in a calcium free solution (EC50 = 11.41 +/- 1.38 micrograms/ml). The modification of the effect by a cyclooxygenase inhibitor (indomethacin), a protein kinase A inhibitor (TPCK), calcium modifying drugs, and drugs interfering with transcription and protein synthesis has been assayed. The effect was unmodified by a 3 microM concentration of indomethacin, (EC50 = 8.77 +/- 1.28 vs 11.41 +/- 1.38 micrograms/ml) and a 5 micrograms/ml concentration of the transcription inhibitor, actinomycin D, (EC50 = 8.23 +/- 2.19 vs 11.41 +/- 1.38 micrograms/ml). The inhibitor of intracellular calcium mobilization TMB-8 (0.1 mM), the Na+/Ca+2 exchanger inhibitor amiloride (0.1 mM), the calcium chelator BAPTA-AM (50 microM), the PKA inhibitor TPCK (10 microM), and the protein synthesis inhibitor cycloheximide (10 micrograms/ml) significantly shifted to the right the dose-response curve of the extract (EC50 = 17.83 +/- 1.87 micrograms/ml, 18.61 +/- 2.50 micrograms/ml, 35.28 +/- 9.13 micrograms/ml, 33.99 +/- 3.07 micrograms/ml, and 27.31 +/- 4.93 micrograms/ml, respectively, vs 11.41 +/- 1.38 micrograms/ml). These results suggest that the effect of the lipidic extract from S. serrulata fruits could be partially due to Na+/Ca+2 exchanger activation and interference with intracellular calcium mobilization, and point to cAMP as a possible mediator. Moreover, protein synthesis seems to be involved in the spasmolytic activity.

Effects of phorbol 12,13-dibutyrate and H-7 in extravascular smooth muscle contraction.

The effect of the activator, phorbol 12,13-dibutyrate (PDB), and the inhibitor, H-7, of protein kinase C (PKC) has been assayed in rat uterus. PDB increases the amplitude of spontaneous contractions of rat uterus and this effect does not occur in the presence of H-7 or nifedipine. PDB did not modify the KCl-induced tonic contraction but H-7 relaxed it, in a concentration-dependent way. PDB inhibited the contraction induced by oxytocin in rat uterus incubated in Ca-free solution and relaxed the tonic contraction induced by oxytocin in this medium. The relaxing effect of PDB on oxytocin-induced contraction was not modified by H-7. Thus H-7 relaxed, in a concentration-dependent way, the tonic contractions induced by oxytocin and vanadate in the rat uterus incubated in Ca-free medium. Our results suggest a dual effect of PDB related to calcium, and a direct and PKC-independent inhibitory effect of H-7.

Mechanisms involved in the effects of phenidone, diclofenac and ethacrynic acid in rat uterus in vitro.

1. The effects of phenidone (P, 10(-4)-10(-3) M), sodium diclofenac (D, 10(-5)-10(-4) M) and ethacrynic acid (E, 10(-5)-10(-4) M), proposed as inhibitors of eicosanoid synthesis, on the contractions of rat uterus induced by several agonists have been studied. 2. P, D and E inhibit the motility induced by oxytocin (4 mU/ml) (IC50: 4.62 x 10(-4), 2.55 x 10(-4) and 2.98 x 10(-5) M, respectively). 3. P (10(-3) M), D (10(-4) M) and E (10(-4) M) also inhibit the contraction induced by methacholine (10(-5) M), prostaglandin F2a (10(-6) M) and CaCl2 (6 mM), and relaxed, in a dose-dependent way, the tonic component of contraction to KCl (60 mM) (IC50: 5.81 x 10(-4), 6.67 x 10(-5) and 7.55 x 10(-5) M, respectively). 4. The CaCl2 (0.1-10 mM) reverted the relaxation of KCl contraction produced by P, but not by D or E. None of the inhibitions on CaCl2 (6 mM) are reverted by Bay K 8644. 5. D and E also relaxed the tonic contraction to vanadate (10(-4) M) in uterus incubated in calcium free solution P, enhances the vanadate-induced contractions.

Involvement of cAMP and beta-adrenoceptors in the relaxing effect elicited by flavonoids on rat uterine smooth muscle.

1. The effect of the flavonoids genistein (3-100 microM), kaempferol (3-60 microM) and quercetin (1-100 microM) on KCl (60 mM)-induced tonic contraction in rat smooth muscle was assayed. In the same way, the modification of these effects in the presence of an inhibitor of protein kinase (PKA) (Rp-cAMPS), an inhibitor of phosphodiesterase (papaverine) and beta-adrenoreceptor blocking agents (propranolol and atenolol) was studied. 2. The flavonoids totally relaxed the KCl-induced tonic contraction (IC50: genistein 20.2 +/- 2.0 microM, n = 11; kaempferol 10.1 +/- 1.6 microM, n = 8; quercetin 13.2 +/- 1.2 microM, n = 8). 3. The incubation with Rp-cAMPS (10 and 100 microM) 30 min prior to KCl shifted the dose-response curve of the flavonoids to the right, increasing their IC50 up to 27.8 +/- 3.8 and 31.9 +/- 7.3 microM, respectively, for genistein; 24.7 +/- 0.2 and 19.6 +/- 4.9 microM, respectively, for kaempferol; 18.8 +/- 2.2 and 18.4 +/- 1.5 microM, respectively, for quercetin. 4. Papaverine (3-100 microM) also relaxed the contraction induced by KCl and this effect was significantly displaced to the right with Rp-cAMPS (10 microM) (IC50 12.1 +/- 2.2 vs. 16.5 +/- 3.1 microM). Papaverine (3 microM) added to the organ bath 15 min before the contractile agent increased the relaxing effect of the flavonoids and significantly decreased their IC50 (genistein 20.2 +/- 2.0 vs. 9.8 +/- 1.4 microM; kaempferol 10.1 +/- 1.6 vs. 6.6 +/- 0.7 microM; quercetin 13.2 +/- 1.2 vs. 7.8 +/- 1.4 microM). 5. The incubation with atenolol (10 microM) did not alter the relaxing effect of the flavonoids. In the same experimental conditions, propranolol (10 microM) did not modify the effect of genistein and kaempferol, but shifted the response curve of quercetin significantly to the right (13.2 +/- 1.2 vs. 17.7 +/- 3.4 microM). 6. The results suggest that genistein, kaempferol and quercetin produced the relaxation of uterine smooth muscle by increasing intracellular cAMP. Beta-adrenoceptors could also be involved in the effect of quercetin.

Non-genomic effects of catecholestrogens in the in vitro rat uterine contraction.

1. The effects of catecholestrogens 2-hydroxyestradiol (2-OH E2, 0.6-30 microM), 4-hydroxyestradiol (4-OH E2, 1-30 microM) and 2-methoxyestradiol (2-MeO E2, 0.6-30 microM) on rat uterine contraction induced by KCl (60 mM), have been assayed. 2. All drugs assayed relaxed the tonic-contraction induced by KCl in a concentration-dependent way. The EC50s were: 4.4 +/- 0.5, 4.2 +/- 0.3 and 8.5 +/- 0.7 microM for 2-MeO E2, 2-OH E2 and 4-OH E2, respectively. This relaxing effect was counteracted by CaCl2 (1-10 mM) but not by the calcium channel agonist Bay k 8644 (1 nM-1 microM). 3. The effect of 2-MeO E2 is not modified by propranolol (1 microM), cycloheximide (35 microM), actinomycin D (4 microM), alpha-difluoromethyl-ornithine (10 mM) or genistein (10 microM). Nor did cycloheximide (35 microM) or actinomycin D (4 microM) modify the relaxing effect of 2-OH E2 and 4-OH E2. Propranolol (1 microM) significantly increased the effect of 4-OH E2 but not the effect of 2-OH E2. 4. Our results suggest that the relaxing effect of catecholestrogens in the rat uterus is a non-genomic effect and could be related to inhibition of extracellular calcium entry.

Calcium-and G-protein-related spasmolytic effects of nonsteroidal anti-inflammatory drugs on rat uterus contractions in vitro.

The effects of the nonsteroidal anti-inflammatory drugs (NSAIDs) acetylsalicylic acid, metamizole, phenylbutazone, indometacin, piroxicam, naproxen, tolmetin, diclofenac, and mefenamic acid on methacholine (10 mumol/l), prostaglandin F2 alpha (1 mumol/l), and KCl (60 mmol/l) induced contractions of isolated rat uterus were assayed. All of these cause a concentration-dependent inhibition of methacholine and prostaglandin F2 alpha-induced contractions with the exception of acetylsalicylic acid, metamizole, and naproxen. All except acetylsalicylic acid and metamizole relaxed in a concentration-dependent manner the tonic contractions induced by KCl. CaCl2 (0.1-10 mmol/l) totally counteracted the relaxant effects of naproxen and tolmetin, but not those of the other NSAIDs. Bay K8644 did not revert the effect of the NSAIDs. Pertussis toxin (50 micrograms/l) did not modify the effect of indometacin, mefenamic acid, and tolmetin, but partially antagonized the effects of diclofenac and naproxen and increased the effect of phenylbutazone and piroxicam. These results suggest that some of the NSAIDs assayed induce smooth muscle relaxation by mechanisms independent of prostaglandin synthesis inhibition, but related to the inhibition of extracellular calcium influx through mechanisms related or unrelated to pertussis toxin sensible G proteins.