KCl-induced Tonic Contraction Research Articles

Relaxant Effects of Piperine and Piperlongumine from Piper longum Fruits on Porcine Coronary Artery.

Since ancient times, Piper longum Linn. fruits have been recognized for exhibiting various effects, including the diaphoretic effects linked to enhanced blood flow. Piperine and piperlongumine coexist in Piper longum Linn. fruits, although the cardiovascular effects of both compounds remain elusive. We investigated their action of piperine and piperlongumine in porcine coronary arteries, comparing them to the Ca2+ channel antagonist diltiazem. Piperlongumine, unlike piperine or diltiazem, concentration-dependently inhibited basal contractile tone in endothelium-denuded coronary arteries. All three compounds inhibit tonic contractions induced by high potassium chloride (KCl) concentrations. The order of relaxation potency indexed by the half-maximal effective concentration (EC50) were as follows: diltiazem > piperlongumine > piperine. These effects were not different between endothelium-intact and -denuded preparations. In endothelial-denuded preparations, pretreatment with these compounds not only inhibited KCl-induced tonic contractions attenuated calcium chloride (CaCl2)-induced ones in a Ca2+-free medium. Histamine-induced phasic contractions in a Ca2+-free medium containing intracellular Ca2+ chelator was completely suppressed by selective inositol trisphosphate receptor antagonist and piperlongumine, whereas piperine or diltiazem do not have the same effect. These findings suggest that piperine and piperlongumine similar to diltiazem cause vasorelaxation by inhibiting both KCl- and CaCl2-induced contractions in coronary arteries, possibly through the inhibition of voltage-dependent Ca2+ channels. Piperlongumine inhibits histamine-induced contractions in a Ca2+-free medium, which is associated with the intracellular Ca2+ signaling pathway, suggesting that the relaxant effect of piperlongumine differs from that of piperine.

In vitro relaxant and spasmolytic effects of essential oil of Pistacia integerrima Stewart ex Brandis Galls

Ethnopharmacological relevancePistacia integerrima J.L. Stewart ex Brandis (Family: Anacardiaceae) galls are used in Indian ethnomedicine for its anti-asthmatic, sedative and spasmolytic properties, however, there are no scientific studies demonstrating its spasmolytic activity. The present investigation deals with the evaluation of relaxant and spasmolytic activities of the essential oil isolated from the galls of Pistacia integerrima J.L. Stewart ex Brandis (EOPI). Materials and methodsIn vitro pharmacological assays were carried out on rabbit jejunum spontaneous contractions, guinea pig ileum. The present investigation studied the relaxation of basal tone of isolated guinea pig ileum by possible involvement of NO, prostaglandins, membrane Na+ channels, potassium channel, enteric nervous system, adrenoceptors, Ca2+ channels. Additional studies were conducted for comparison of the relaxant effects of EOPI on CaCl2 induced contraction in calcium free tyrode solution, effect on nifedipine insensitive component of ACh-induced contraction and on the contractile machinery to intracellular [Ca2+] on isolated guinea pig ileum. ResultsEOPI at non-relaxing dose potentiated the isoprenaline induced relaxation of rabbit jejunum. EOPI (50µg/mL) exhibited 28% relaxation of basal tone of 60mMK+ induced contraction which is unaltered by preincubation with 0.5mM hexamethonium, 0.5µM Tetrodotoxin, 1µM indomethacin, and 100µML-NG-Nitroarginine Methyl Ester (L-NAME). EOPI inhibited Ca2+ induced contraction of isolated guinea pig ileum in Ca2+ free medium. EOPI (10µg/ml) potentiated the reversal of a KCl-induced tonic contraction has been observed in Ca2+ free medium. ConclusionThe present investigation reinforces the use of Pistacia integerrima Stewart ex Brandis as antispasmodic in folk medicine. Moreover, it is demonstrated the involvement of β- adrenoceptors and calcium channels in this activity, but not the participation of nicotinic receptors, Na+ channels, prostaglandins or nitric oxide.

Potent Inhibition of Arterial Smooth Muscle Tonic Contractions by the Selective Myosin II Inhibitor, Blebbistatin

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC(50) = 0.5-5 microM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC(50) approximately 80 microM). We found that blebbistatin potently (IC(50) approximately 3 microM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC(50) approximately 5 microM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited beta-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC(50) approximately 20 microM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.

The potency of KUL-7211, a selective ureteral relaxant, in isolated canine ureter: comparison with various spasmolytics

We compared the potency of a selective ureteral relaxant KUL-7211 (beta(2)/beta(3)-adrenoceptor agonist; (-)-2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenyloxy]acetic acid) with those of various spasmolytics on contractions in isolated canine ureteral preparations. Drug effects were evaluated on the tonic contraction induced by KCl (80 mM) and on spontaneous, 1x10(-5) M phenylephrine-, and 1x10(-6) M PGF(2alpha)-induced rhythmic contractions in isolated canine ureteral preparations using a functional experimental technique. The potencies (pD(2) value) of the following drugs were compared: KUL-7211, tamsulosin (an alpha(1A/1D)-adrenoceptor antagonist), prazosin (an alpha(1)-adrenoceptor antagonist), verapamil (a Ca(2+)-channel blocker), butylscopolamine (a nonselective muscarinic antagonist), and papaverine (a phosphodiesterase inhibitor). The rank order of relaxing potencies against KCl-induced tonic contraction was KUL-7211 (6.60)>tamsulosin(5.90)>verapamil(5.70)>papaverine(4.88)>prazosin (4.54). The rank order of potencies for reductions in spontaneous rhythmic contractions was KUL-7211 (6.80)>verapamil(6.12)>papaverine(5.05). Conversely, high concentrations of the two alpha-adrenoceptor antagonists (tamsulosin and prazosin) and of butylscopolamine enhanced the spontaneous contractions, although at low concentrations (up to 1x10(-6) M) they had no significant effects. For suppression of spasmogen-induced rhythmic contractions, the rank order of potencies was, against phenylephrine-induced contractions: KUL-7211 (6.95)>tamsulosin(6.26)>prazosin(5.68)>verapamil(5.64)>papaverine (5.03), and against PGF(2alpha)-induced contractions: KUL-7211 (7.05)>verapamil(6.70)>papaverine (5.27). Our results suggest that in dogs, the beta(2)/beta(3)-adrenoceptor agonist KUL-7211 is the most efficacious ureteral relaxant among the spasmolytics tested against various contractions. Possibly, KUL-7211 might be useful for promoting stone passage and relieving ureteral colic in urolithiasis patients.

Smooth muscle relaxant effect of kaurenoic acid, a diterpene from Copaifera langsdorffii on rat uterus in vitro.

The effect of kaurenoic acid, a diterpene isolated from the oleo-resin of the popular medicinal plant Copaifera langsdorffii (Leguminaceae), was analysed on rat uterine muscle responsiveness to various drugs in vitro. Cumulative concentration-response curves to acetylcholine and oxytocin were obtained before and after incubation of uterine segments with up to 160 microm of kaurenoic acid. The maximal contractile response (E(max)) evoked by these agonists was inhibited by kaurenoic acid in a concentration-related manner; at 160 microm, kaurenoic acid depressed the E(max) of oxytocin and acetylcholine by 83% and 91%, respectively. The relaxation caused by kaurenoic acid on oxytocin-induced contraction was unaffected in the presence of tetraethyl ammonium, a compound that blocks the calcium activated potassium channels. It was partially reversed by glibenclamide (10(-5) m), an ATP-sensitive potassium channel blocker. Also, kaurenoic acid at 160 microm concentration was found to inhibit significantly the CaCl(2)-evoked contractile responses in a medium of high potassium and zero calcium. Furthermore, kaurenoic acid was found to relax the sustained tonic contraction induced by acetylcholine, oxytocin, BaCl(2) and KCl in a concentration-dependent way. However, KCl-induced tonic contraction was only weakly inhibited by kaurenoic acid. These data indicate that the diterpene, kaurenoic acid, exerts a uterine relaxant effect acting principally through calcium blockade and in part, by the opening of ATP-sensitive potassium channels.

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

The effect of kaempferol on KCl (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 (α-difluoromethyl-ornithine, DFMO), as well as a polyamine, spermine, have been assayed. Kaempferol (3 to 60 μM) induced concentration-dependent relaxation on KCl-induced tonic contraction (IC 50: 10.1 ± 1.89 μM). This relaxing effect was antagonized (p<0.05) by Rp-cAMPS (10 μM), TPCK (3 μM), DDA (100 μM), actinomycin D (4 and 12 μM), cycloheximide (100 μM), DFMO (10 mM), actinomycin D (12 μM) plus TPCK and actinomycin D (12 μM) 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.

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 CaCl 2(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 μM), cycloheximide (300 μM), TPCK (3 and 10 μM), Rp-cAMPS (30 μM), DDA (100 μM) and H-7 (1 μM). None of these modified the relaxing effect of mifepristone. Incubation with drugs that interfere with cGMP such as a nucleotide analogue DDG (100 μM), a soluble guanylyl cyclase inhibitor ODQ (1 μM) and an inhibitor of protein kinase G 8pCPTcGMPS (1 μM) significantly modified the effect of mifepristone, increasing its IC 50.

Effects of ovarian hormones on beta-adrenergic receptor-mediated relaxation in the female rabbit bladder.

The effects of ovarian hormones on beta-adrenergic receptor-mediated responses in female rabbit detrusor smooth muscles were investigated. Ovariectomized mature female New Zealand white rabbits were untreated or treated with estrogen and/or progesterone for 2 weeks. The contractile responses to carbachol and KCl in the detrusor strips were not significantly different in all groups. As compared with dobutamine and GS-332, isoproterenol and procaterol significantly relaxed the detrusor strips derived from all groups on KCl-induced tonic contractions. Combined with estrogen treatment, isoproterenol, procaterol and GS-332 caused a significant increase in this muscle relaxation. Furthermore, estrogen treatment caused a significant increase in relaxation as a result of forskolin and the cyclic AMP (cAMP) production that was induced by isoproterenol, procaterol and GS-332. However, estrogen treatment did not affect the relaxant response to dibutyryl cyclic AMP. Progesterone treatment did not affect beta-adrenergic receptor-mediated responses. These results suggest that estrogen treatment causes the increased relaxant responses mediated by beta2- and beta3-adrenergic receptor subtypes, which may be related to the increased cAMP content in female rabbit detrusor smooth muscles.

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.

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

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.

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 (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.

Mechanisms of Spermine‐ and N1‐Acetylspermine‐induced Relaxation on Rat Uterine Smooth Muscle In‐vitro

The effect of the natural polyamines, spermine and N1-acetylspermine, on rat uterine smooth muscle and cAMP phosphodiesterase activity has been studied to determine the actions that contribute to their inhibitory effect on smooth muscle. Both drugs produce concentration-dependent relaxation of KCl-induced tonic contraction in rat uterus, which could be counteracted by CaCl2 (0·1–3·0 mM). 1(5 Isoquinolinyl-sulphonyl)-2-methyl-piperazine (1 μM) and N-p-tosyl-l-phenylalanine-chloromethyl-ketone (3 μm), inhibitors of protein kinase C and A, respectively, did not modify the relaxant effect of spermine or N1-acetylspermine. Basal activity and calmodulin-stimulated activity of cAMP phosphodiesterase was inhibited in a concentration-dependent way by spermine, but not by N1-acetylspermine. These results suggest that spermine and N1-acetylspermine produce their spasmolytic effect on rat uterus through a calcium-antagonistic action. The inhibition of phosphodiesterase and calmodulin does not appear to be involved in the relaxant effect induced by spermine, as it occurs at different concentrations and is unmodified by the protein kinase A inhibitor.

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 μM), forskolin (1–60 μM), papaverine (1–100 μM), vinpocetine (1–100 μM), rolipram (0.1–1 mM), Sp-cAMPS (10–300 μM), 8-BrcAMP (10 μM-1 mM) and 8-BrcGMP (3 μM-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 μM) 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.

Age-dependent Alterations in beta-adrenergic Responsiveness of Rat Detrusor Smooth Muscle

The relaxant effects of norepinephrine (NE, 10 sup -7 to 10 sup -4 M.) and isoproterenol (ISO, 10 sup -9 to 10 sup -4 M.) on maximal KCl-induced tonic contractions and the relaxant effects of ISO on contractions induced by electrical field stimulation (EFS) were measured in detrusor muscle strips obtained from 22-25 day, 90-95 day and 22-month-old male Fischer 344 rats. The maximum relaxant response to NE and ISO on KCl-induced tonic contractions decreased significantly with increasing age. The ED 50 values for ISO, but not NE, increased with age. The maximum relaxant response to ISO on EFS-induced contractions also was reduced significantly in the old bladders. The relaxation effects of forskolin (10 sup -6 to 3 X 10 sup -5 M.), dibutyryl cyclic AMP (DBcAMP, 10 sup -4 to 3 X 10 sup -3 M.) and cholera toxin (10 microgram/ml.) were examined on maximal KCl-induced contractions of the muscle strips obtained from the three age groups. The relaxant responses to forskolin decreased significantly with increasing age, whereas DBcAMP relaxed the muscle strips from the three age groups equally. Cholera toxin (10 microgram) attenuated KCl-induced phasic contractions, and this effect was impaired in the aged rat detrusor. The density of beta-adrenergic receptors, as determined by radioligand binding with [sup 125 I]iodopindolol ([sup 125 I]-PIN) decreased with increasing age. These data demonstrate an age-related decrease in the responsiveness of the bladder detrusor to beta-adrenergic stimulation that may be related to the decreased density of beta-adrenergic receptors and decreased cyclic AMP (cAMP) production.

Effects of androgens on isolated rat uterus

The 5α- and 5β-dihydrotestosterone (DHT) androgens relax KCl-induced tonic contraction in rat uterus, in a dose-dependent way. The 5α and 5β-DHT relaxing effect is counteracted by CaCl 2 (0.1–10 mM). The 5α-DHT, but not the 5β-DHT, effect was reduced by cycloheximide (10 μg/ml) and by actinomycin D (5 μg/ml). Flutamide at 10 −6 M shifted the effect of 5α-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 5α-DHT and 5β-DHT in rat uterus contraction but that intracellular and genomic actions play a part in the relaxing effect of 5α-DHT.

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.

Effects of antiandrogens and progesterone on isolated rat uterus

1. 1. The effect of progesterone (P, 6 × 10 −6−6 × 10 −5 M) and the antiandrogens cyproterone acetate (CAP, 10 −7–10 −5 M), flutamide (F, 10 −6−6 × 10 −5 M) and spironolactone (S, 10 −6−6 × 10 −5 M) on the KCl-induced tonic contraction of the isolated rat uterus have been assayed. 2. 2. The antiandrogens relaxed, in a dose-dependent way, the KCl-induced contraction (EC 50, 2.804 ± 0.506 × 10 −6; 1.671 ± 0.308 × 10 −5 and 3.042 ± 0.14 × −5 M , respectively for CPA, S and F) P also relaxed the KCl-induced contraction (EC 50, 2.436± 0.524 × 10 −5 M). 3. 3. CaCl 2 (0.1–10 mM) counteracted the relaxing effect of CPA, S. F, and P respectively, up to 100, 80.63, 60.66 and 90.57%. 4. 4. The 17-OH-progesterone derivative CPA, but not S or F, reduces at small doses (6 × 10 −8 M), but not at higher concentrations (6 × 10 −7−6 × 10 −6 M), the relaxing effect of progesterone.