Role of the serotonergic pathway in uterotonic activity of Ananas comosus (L.) Merr. - An in vitro and in vivo study.

BACKGROUND: Ananas comosus (L.) Merr. has been used as a traditional medicine in inducing abortion in many countries. Our previous in vitro experiments showed that the aqueous fraction (F4) of A. comosus extract stimulated the rat and human uterine contractions. PURPOSE: The aim of this study was to identify the bioactive compound and further investigate the molecular mechanism of F4 induced contraction and the in vivo uterotonic effect of F4. MATERIALS AND METHODS: Organ bath studies were employed to compare the stimulatory effect of F4 in non and late pregnant uterine tissue followed by isolation of protein from late pregnant uterine tissue for the western blot analysis. The PhysioTel transmitter was implanted in pregnant SD rats to measure the changes in intrauterine pressure (IUP). Analyses of the crude extract and active principle in F4 was performed using LC-HRMS. RESULTS: Ripe F4 in a similar manner as serotonin produced a greater stimulatory response in late pregnant than non-pregnant uterine tissue without significant change in potency; ripe F4 also increased ERK phosphorylation which eventually led to a significant increase of the final product, MMP-13. In pregnant rats (E18), oral ripe F4 (1.5 g.100 g-1 body weight) and ergometrine (1 mg) did not stimulate the uterine contraction probably due to the low level of estradiol and as a consequence low 5-HT receptors at the time of administration. In contrast, in postpartum rats, oral administration of F4 and ergometrine produced a significant increase in maximal IUP to 4.3 and 4.9 folds of basal IUP respectively. Contrary to the folklore use, unripe F4 did not stimulate the uterine activity during pregnancy and postpartum. Bioassay guided fractionation identified serotonin as a major bioactive compound in ripe F4. CONCLUSIONS: Our data clearly indicate that the uterotonic effect of ripe F4 is mediated via the serotonergic pathway and suggest that serotonin rich diet may increase the peripheral serotonin and implicate in diverse physiological functions, including uterine motility.

Potent tocolytic activity of ethyl acetate fraction of Ananas comosus on rat and human uteri.

The aim of this study was to investigate the tocolytic properties of Ananas comosus extract in rat and human uterine tissue in vitro and in the rat in vivo. Organ bath technique was employed to perform functional studies in vitro. The PhysioTel transmitter was implanted in SD rats to measure the changes in intrauterine pressure (IUP) in vivo. Analyses of F2 was performed using LC-HRMS. F2 produced a non-selective inhibitory response on oxytocin, prostaglandin F2α, acetylcholine and KCl. The inhibitory activity of F2 on oxytocin-induced contraction was not attenuated by propranolol, TEA, glibenclamide and indomethacin. Nω-Nitro-L-arginine, a nitric oxide synthase inhibitor, suppressed the maximal tocolytic activity of F2 by 25%. DIDS, an inhibitor of chloride channels, appeared to suppress the relaxant effect of F2. F2 suppressed the oxytocin-induced contraction in Ca2+ free solution. The in vivo tocolytic activity of F2 and ritodrine were observed in non-pregnant rats during the estrous stage by suppressing the frequency and amplitude of IUP peaks following intrauterine administration. Chemical analysis confirmed the involvement of citric acid in the tocolytic activity of F2. However, another less polar fraction is essential to accompany citric acid to produce such potent inhibitory response of F2. It is likely that F2 exerted tocolytic activity by multiple mechanisms, including antagonizing L-type Ca2+ channels, interfering with the intracellular Ca2+ release mechanism and releasing nitric oxide. F2 would be a promising candidate to develop as a tocolytic agent.

Endothelial colony forming cells from human umbilical cord blood improved severe erectile dysfunction in obese type II diabetic rats.

AIM: To investigate the effect of intracavernous injection of human umbilical cord blood derived endothelial colony forming cells (HUCB ECFCs) on erectile dysfunction (ED) in Zucker Diabetic Fatty (ZDF) rat model. METHODS: Erectile function was assessed by cavernous nerve electrostimulation in ZDF rats aged 20-28 weeks. Following confirmation of severe ED at the age of 28 weeks, 21 ZDF rats were randomly assigned to three experimental groups: 1 million ECFCs, 2 million ECFCs, and phosphate buffered saline (PBS). Four weeks after intracavernous injection, the efficacy of ECFCs was quantified by intracavernous pressure (ICP) measurement, Masson's trichrome staining, immunohistologic and immunoblot analyses and TUNEL assay. KEY FINDINGS: Intracavernous ECFC administration improved ICP in a dose-dependent manner in comparison to the age-matched PBS group. Functional improvement in ICP was accompanied by a significant restoration of the cavernosal endothelial and smooth muscle cell content and cavernosal nerve function. The percentage eNOS and nNOS positive cavernosal cells, and their respective protein expression levels and nNOS positive cells in the dorsal penile nerve in 2 million ECFCs treated groups were significantly higher than the PBS group. TUNEL stain quantification showed a significant decrease in cavernosal apoptosis following ECFC treatment. SIGNIFICANCE: The results are expected to provide a scientific basis to further study the clinical application of HUCB ECFCs in ameliorating ED in human. CONCLUSIONS: HUCB ECFCs significantly improved severe ED in ZDF rats through improvement of the nerve and endothelium function and restoration of smooth muscle in the cavernosum by overcoming the cavernosal apoptosis.

Mechanisms of direct relaxant effect of sildenafil, tadalafil and vardenafil on corpus cavernosum.

Sildenafil, tadalafil, vardenafil and verapamil induced concentration-dependent relaxation of the rabbit corpus cavernosum muscle precontracted with noradrenaline. The maximal relaxation (%) at 20 microM was 61.4 +/- 6.9, 32.4 +/- 5.4, 100.0 +/- 5.5 and 86.6 +/- 5.1 (n = 5 each) respectively. Pre-incubation of cavernosal muscle strips with N(omega)-nitro-L-arginine or guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but not adenylate cyclase inhibitor, cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine] (MDL12330A) culminated in only a 20-30% reduction in muscle relaxant action of the 3 phosphodiesterase inhibitors. This suggests that another mechanism of relaxation independent of nitric oxide-cGMP or cAMP pathway was involved. Higher concentrations of sildenafil (100 microM) and vardenafil (10 and 100 microM) produced non-competitive antagonism of noradrenaline-induced contraction characterized by reduced maximal effect. In contrast, tadalafil was devoid of significant effect on noradrenaline. On K(+)-depolarized tissues, sildenafil was as potent as vardenafil whereas tadalafil was the least effective in relaxing K(+)-induced tone. The maximal relaxation (% of K(+)-induced tone) at 20 microM sildenafil, tadalafil and vardenafil was respectively 84.1 +/- 6.5, 9.0 +/- 19.9, and 88.9 +/- 6.2 (n = 5 each). In addition, verapamil, sildenafil and vardenafil were more efficacious than tadalafil in reversing tonic contractions by Ca(2+) channel activator, 1,4,dihydro-2,6-dimethyl-5-nitro-4-[2(triflouromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (BAY K-8644). These results indicate that vardenafil and sildenafil possess direct muscle relaxant potential possibly via inhibiting Ca(2+) influx through both receptor-operated and voltage-dependent Ca(2+) channels whereas tadalafil appears capable of inhibiting receptor-operated transmembrane Ca(2+) entry only.

Investigation of uterotonic properties of Ananas comosus extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: In folklore medicine Ananas comosus (pineapple) is reputed to act as an abortifacient and in expectant women as a means of inducing labor. Several reports have claimed abortifacient property of A. comosus fruit (ripe or unripe). Ripe fruit has been used orally as traditional medicine in inducing abortion in Kerala state of India while the juice of unripe fruit was used for abortion in Bangladesh. However, scientific evidence supporting the efficacy of pineapple extracts in inducing uterine contractions is clearly lacking. AIM OF THE STUDY: This study investigated the pharmacological effects of different fractions of pineapple extract with a range of maturities to identify the most potent uterotonic fraction. MATERIALS AND METHODS: The ethanolic crude extracts of pineapple (edible part) were prepared and fractionated through a series of liquid-liquid partitions. Fractions were separately tested on isolated uterine muscle from pregnant SD rats and human pregnant myometrium, which were cut into strips along the longitudinal axis of uterus. The strips were mounted vertically in organ baths (37°C) and exposed to cumulative addition of fractions (0.1-10mgml-1), serotonin (0.05-5µM) and different inhibitors to delineate the mechanism of action of the active ingredients of the extract. RESULTS: Aqueous fraction (F4) possesses uterine stimulant property which was blocked by verapamil but unaffected by indomethacin, prazosin and atosiban. Notably, ketanserin (10µM) diminished the maximal contractile response induced by both F4 and 5HT by 74.3% and 92.1% respectively. CONCLUSIONS: These results may indicate the presence of 5HT or 5HT-like compound(s) and serotonergic pathways may contribute to the uterotonic activity of pineapple extract.

Effects of propofol on guinea pig respiratory smooth muscle.

The effects of propofol on the tone of guinea pig respiratory smooth muscle was studied both in vitro and in vivo. In vitro, the activity of propofol on tracheal smooth muscle was investigated using a force displacement transducer for isometric tension responses. Isoproterenol was used as the control. Concentration-response curves to propofol and isoproterenol were obtained using a cumulative dose schedule. Propofol (0.32-10.24 µg·ml-1) relaxed the tracheal smooth muscle in a concentration-dependent manner, but was less potent than isoproterenol (equipotent molar ratio 29 000∶1). This effect of propofol was not affected by prior administration of atropine, propranolol, prazocin, or yohimbine, and it did not appear to be mediated via calcium antagonism. The solvent for propofol (10% intralipid) had no effect on the tracheal smooth muscle in vitro. The in vivo study measured the effect of propofol on lung pressure in deeply anesthetized guinea pigs using histamine induced bronchoconstriction. Propofol (1-4.5 mg·kg-1, i.v.) exhibited neither relaxant nor constrictor effects. It is possible that the effects of propofol observed in vitro are due to nonspecific action, while the finding of no effect in vivo could be due to different tissue sensitivity to propofol, i.e., tracheal smooth muscle may be more responsive than bronchial smooth muscle. Propofol does not seem to have any deleterious effects on airway smooth muscle.

Characterizing behavior of corpus cavernosum in chloride-free condition.

OBJECTIVE: To elucidate the role of chloride currents in erectile function through characterizing the behavior of corpus cavernosum (CC) in chloride-free (Cf) medium, which has not been evaluated before. METHODS: Isolated rabbit CC strips were suspended in thermo-regulated organ baths containing oxygenated Tyrode for isometric tension recording. Cf Tyrode was prepared by substituting sodium chloride, calcium chloride, and potassium chloride (KCl) with equivalent molar concentrations of sodium acetate, calcium acetate, and potassium acetate salts. Resting cavernosal tone and contractions by noradrenaline, histamine, and KCl were assessed in Cf Tyrode with or without chloride channel blockers, niflumic acid (NFA), and anthracene-9-carboxylic acid (A9C). RESULTS: Withdrawal of extracellular chloride caused myogenic contractions in the unstimulated CC strips (n = 18). In addition, peak contractions by noradrenaline (n = 14) and histamine (n = 13) were augmented in Cf buffer by 47.2 ± 5.9% and 85.4 ± 13.2%, respectively (P <.05), whereas KCl contractions were not significantly altered (17.6 ± 4.6%; n = 7). Interestingly, Cf buffer exerted opposing effects, potentiation and reduction, respectively, on the plateau phase of contractions mediated by noradrenaline and histamine. The stimulatory effect of Cf buffer on the intrinsic myogenic tone was diminished by NFA (30 µM), and A9C (300 µM-1 mM). NFA (30-100 µM), however, specifically reduced the plateau phase without significantly modifying the peak contraction of noradrenaline in Cf buffer. CONCLUSIONS: These results reiterate the importance of chloride currents as a mechanism underlying the maintenance of penile cavernosal tone. Thus, chloride channel could be an effective alternative target to regulate penile erection.

Effect of sildenafil and rolipram on adrenergic responses in isolated human and monkey corpus cavernosum.

OBJECTIVE: Evaluate and compare effects of phosphodiesterase inhibitors (PDEIs), sildenafil and rolipram, on adrenergic contractile responses of human and monkey cavernosal smooth muscle. METHODS: Human penises were obtained from patients undergoing gender reassignment surgery. Isolated human and monkey corpus cavernosum (CC) strips were suspended in tissue bath chambers for isometric tension experiments. The effects of the drugs on precontracted monkey and human CC and neurogenic contractions in human CC were investigated. RESULTS: Both sildenafil and rolipram induced concentration-dependent relaxations of human and monkey CC strips precontracted with noradrenaline (NA). The IC(50) values, determined by reverse regression for nitroglycerin (NTG), isoprenaline, and sildenafil in monkey CC, were, respectively, 1.5+/-0.9x10(-7) M, 3.7+/-0.6x10(-6) M, and 1.7+/-0.7x10(-5) M. Similarly, in human CC muscle, sildenafil was weaker than NTG as a muscle relaxant. Sildenafil, 1.5 microM, reduced neurogenic contractions in human CC due to stimulation of predominantly adrenergic nerves. The suppressant effect of sildenafil on adrenergic transmission was attenuated in CC strips pretreated with N omega-nitro-L-arginine and overcome with a higher stimulus frequency or tetraethylammonium. Rolipram partially inhibited adrenergic excitatory response but without significantly affecting NA-induced contraction. CONCLUSIONS: Sildenafil and rolipram induced concentration-dependent reversal of human and monkey CC tone mediated by NA. Both PDEIs attenuated contractile adrenergic response of human CC to electrical stimulation. The results also underline the importance of the cyclic adenosine monophosphate-dependent signalling pathway in regulating the tone. PDE4 inhibition in CC is an additional mechanism for erection and potential therapeutic adjunct.