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Background: Harungana madagascariensis Lam. ex Poir. (Hypericaceae) leaf extract is used by some communities in the Niger Delta to treat diarrhoea, ulcers, and wounds. Objectives: This study investigated the antispasmodic, antimotility and gastroprotective properties of methanol:dichloromethane (1:1) extract of H. madagascariensis leaves (HME). Materials and Methods: The antispasmodic activity was evaluated in vitro using actions on contractions of guinea pig ileum and rabbit jejunum provoked by spasmogens. The actions of HME on gastrointestinal transit was assessed in vivo using normal defaecation and charcoal meal transit time tests in rodents. The actions of HME on gastric ulcers produced by ethanol and indomethacin were investigated. The HME was also subjected to phytochemical analysis and acute toxicity tests. Results: The HME suppressed contractions of isolated rabbit jejunum and guinea pig ileum elicited by histamine and acetylcholine. The extract elicited significant (P<0.05) reduction of normal defaecation (12.50 - 100%) and gastrointestinal propulsion of charcoal meal in mice (17.60 - 43.08%). Additionally, the extract significantly (P<0.05) prevented both ethanol-and indomethacin-induced stomach ulcers. An oral LD50 >5000 mg/kg in mice was obtained by an acute toxicity assay on HME. Conclusion: The findings showed that the leaf of H. madagascariensis has gastroprotective, antispasmodic, and antimotility properties. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Seed kernel of the plant Ceasalpinia bonducella Linn (Caesalpiniacaeae) are used for the treatment of asthma in folk medicine and ancient books. Aim of study To assess the pharmacological efficacy of the plant in asthma and to confine and describe the synthetic constituents from the seeds that are in charge of the action. Material and methods The viability of petroleum ether, ethanol extract and ethyl acetate fraction from ethanol extract of C. bonducella seeds were screened for the treatment of asthma by various methods viz. effect of test drug on clonidine and haloperidol induced catalepsy, milk-induced leukocytosis and eosinophilia, mast cell stabilizing activity in mice and studies on smooth muscle preparation of guinea pig ileum (in-vitro). Column chromatography of active extract was done to pinpoint the active compound followed by structure elucidation by FTIR, GCMS and NMR spectroscopic methods. Results Ethyl acetate fraction from ethanol extract of C. bonducella seeds exhibited antihistaminic activity at the dose of 50 and 100 mg/kg, inhibited clonidine-induced catalepsy but not haloperidol-induced catalepsy. Ethyl acetate fraction from ethanol extract significantly inhibited increased leukocyte and eosinophil count due to milk allergen and showed maximum protection against mast cell degranulation by clonidine. The results of guinea pig ileum indicated that the compound 2 methyl, 1 hexadecanol isolated from ethyl acetate fraction of ethanol extract relaxed significantly the ileum muscle strips pre-contracted by which suggests the involvement of β 2 -agonists on the relaxation of the tissue. All the results are dose dependent. Active ethyl acetate fraction from ethanol extract showed presence of anti-asthmatic compound, 2-methyl, 1-hexadecanol. Conclusion The ethyl acetate fraction from ethanol extract of seeds of the plant C. bonducella can inhibit parameters linked to asthma disease. [ABSTRACT FROM AUTHOR]

In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC50 = 7.0 ± 1.9 × 10−5 M), histamine (IC50 = 1.3 ± 0.3 × 10−4 M) and serotonin (IC50 = 8.0 ± 1.4 × 10−5 M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD′2 = 4.48 ± 0.08), shifting the curves to the right with Emax reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC50 = 9.0 ± 0.9 × 10−5 M) and carbachol (EC50 = 4.6 ± 0.7 × 10−5 M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca2+ influx through voltage-gated calcium channels (CaV), since caulerpine slightly inhibited the CaCl2-induced contractions in depolarizing medium without Ca2+, shifting the curves to the right and with Emax reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca2+ influx through CaV. However, other mechanisms are not discarded.

The protein-containing extracts prepared from the venom ducts of Conus austini, Conus spurius and Polystira albida caused a concentration-dependent inhibition of spontaneous contractions in guinea pig ileum. The most potent extract was obtained from P. albida venom ducts (IC50 = 0.11 ± 0.02 µg protein/mL). The three extracts produced a moderate inhibition of contractions elicited by acetylcholine (ACh 1 µM), suggesting the presence of anticholinergic compounds. The contractile response elicited by nicotine (10 µM) was significantly reduced by the extracts prepared from the ducts of C. austini and P. albida, which indicates that the venom produced by these species contains toxins that target neuronal nicotinic receptors. All three extracts significantly inhibited contractions evoked by histamine (0.5 µM), particularly those from C. spurius and P. albida. These findings reveal the presence of antihistaminergic compounds not previously described in any conoidean venom. Finally, we found that only the extract prepared from C. spurius ducts decreased KCl (60 mM)-induced contractions, indicating that the venom of this snail contains compounds that block voltage-dependent Ca2+ or Na+ channels.

Background: It has been recently shown that essential oil of Nigella sativa seeds and its major constituent, thymoquinone (TQ), possess relaxant activity on isolated trachea and ileum. Several mechanisms have been suggested to explain the smooth muscle relaxant effects of the essential oil of Nigella sativa seeds. But, only in one study, it has been shown that TQ exerts its relaxant activity probably through the inhibition of histaminergic and serotonergic receptors. Objective: This study was carried out in order to further evaluating the mechanism(s) of muscle relaxant activity of TQ. Methods: Tension recording technique using an isometric transducer connected to a physiograph in an organ bath set up was used. The spontaneous contractions of isolated ileum and contractile responses evoked by acetylcholine (ACh), histamine (His), potassium chloride (KCl), and repetitive electrical field stimulation (EFS), were recorded. ACh (10 M), His (100 M), KCl (10 mM), and EFS (20 Hz, 0.2 ms, 130 V), were applied before and after adding TQ (10, 20, 40, 80, 100 M). Moreover, in order to evaluate the role of Ca2+-channels in TQ-induced relaxation of ileum, the amplitude of contractions evoked by EFS were recorded in the presence and absence of TQ (80 M), in Ca2+-free Krebs solution, as well as following adding of cumulative concentrations of CaCl2 (0.1, 0.2, 0.4, 1, 2 mM). Results: The results showed that TQ at the minimum concentration of 20 M decreased the amplitude of spontaneous contractility (p

The management of hypertension poses a challenge in both the developing and developed countries. However, the disease is more prevalent in the developing countries where poverty, illiteracy and poor nutrition combine to exacerbate the condition. The study was carried out to evaluate the in vitro and in vivo hypotensive properties of the ethanolic extract of Moringa oleifera Lam. (Moringaceae) seeds. The oscillographic technique was employed on guinea pig ileum and a live cat to evaluate the antihypertensive properties of the extract. In the in vitro relaxation studies, the extract induced contraction on the ileum in a dose-dependent manner; extract doses of 1, 2 and 4 mg mL-1 induced responses (mm) of 2.32 ± 0.46, 4.50 ± 0.46 and 7.52 ± 0.56 respectively, compared to the control value of 1.42 ± 0.42 induced by 2µg/ml of acetylcholine. In the in vivo studies, the extract significantly reduced the mean arterial blood pressure (mm Hg) of an anaesthesized normotensive cat from a control value of 116 ± 6.33 (when normal saline was administered) to 80 ± 3.30, 50 ± 3.70 ± 3.10 and 52 ± 6.60 at doses of 10, 20 and 40 mg ml-1 respectively. These findings corroborate the use of M. oleifera seed as an antihypertensive agent in ethno-medicine.

Previously, we demonstrated that caulerpine has spasmolytic effect on guinea pig ileum. The aim of this study was to investigate pathways of its spasmolytic action. We test caulerpine against phasic contractions induced by carbachol in the circular layer of guinea pig ileum and this alkaloid did not inhibit these contractions, indicating that caulerpine did not interfering with the mobilisation of Ca2+ from intracellular stores. Additionally, the spasmolytic effect of caulerpine did not involve K+ channels. Furthermore, we observed that α2-adrenergic receptors were not involved in the spasmolytic effect of caulerpine, since the relaxation curve induced by caulerpine was not shifted in the presence of yohimbine (α2-adrenergic antagonist). However, in the presence of propranolol (β-adrenergic antagonist), the relaxation curve induced by caulerpine was right-shifted, resulting in a fivefold increase in EC50. Thus, a possible mechanism for the spasmolytic action of caulerpine is the activation of β-adrenergic receptors. [ABSTRACT FROM AUTHOR]

In the present study, methanolic extract of tuber of Stephania glabra was evaluated for H1-bloker activity by employing in vitro screening models of guinea pig ileum and goat tracheal chain preparation. Goat isolated trachea and guinea pig ileum contracted to histamine in a dose-dependent manner while chlorpheniramine blocked this effect. The methanolic extract produced significant dose-dependent H1-receptor antagonist activity by blocking histamine-induced contraction.

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of ³H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 µM) and up to 10 min of treatment, crotoxin decreased ³H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 µM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on ³H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits ³H-choline uptake with high affinity (EC25 = 10 ± 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

Background: Xylopia frutescens Aubl. (embira, semente-de-embira or embira-vermelha), is used in folk medicine as antidiarrheal. The essential oil from its leaves (XF-EO) has been found to cause smooth muscle relaxation. Thus, the aim of this study was to investigate the spasmolytic action by which XF-EO acts on guinea pig ileum. Methods: The components of the XF-EO were identified by gas chromatography-mass spectrometry. Segments of guinea pig ileum were suspended in organ bath containing modified Krebs solution at 37 °C, bubbled with carbogen mixture under a resting tension of 1 g. Isotonic contractions were registered using kymographs and isometric contractions using force transducer coupled to an amplifier and computer. Fluorescence measurements were obtained with a microplate reader using Fluo-4. Results: Forty-three constituents were identified in XF-EO, mostly mono- and sesquiterpenes. XF-EO has been found to cause relaxation on guinea pig ileum. The essential oil inhibited in a concentration-dependent manner both CCh- and histamine-induced phasic contractions, being more potent on histamine-induced contractions as well as antagonized histamine-induced cumulative contractions in a non-competitive antagonism profile. XF-EO relaxed in a concentration-dependent manner the ileum pre-contracted with KCl and histamine. Since the potency was smaller in organ pre-contracted with KCl, it was hypothesized that XF-OE would be acting as a K+ channel positive modulator. In the presence of CsCl (non-selective K+ channel blocker), the relaxant potency of XF-OE was not altered, indicating a non-participation of these channels. Moreover, XF-EO inhibited CaCl2-induced cumulative contractions in a depolarizing medium nominally without Ca2+ and relaxed the ileum pre-contracted with S-(-)-Bay K8644 in a concentration-dependent manner, thus, was confirmed the inhibition of Ca2+ influx through Cav1 by XF-EO. In cellular experiments, the viability of longitudinal layer myocytes from guinea pig ileum was not altered in the presence of XF-OE and the Fluo-4-associated fluorescence intensity in these intestinal myocytes stimulated by histamine was reduced by the essential oil, indicating a [Ca2+]c reduction. Conclusion: Spasmolytic action mechanism of XF-EO on guinea pig ileum can involve histaminergic receptor antagonism and Ca2+ influx blockade, which results in [Ca2+]c reduction leading to smooth muscle relaxation. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the pharmacological activity of Pronetupitant, a novel compound designed to act as prodrug of the NK 1 antagonist Netupitant. In receptor binding experiments Pronetupitant displayed high selectivity for the NK 1 receptor. In a calcium mobilization assay performed on CHO NK1 cells Pronetupitant (100 nM, 15 min preincubation) behaved as an NK 1 antagonist more potent than Netupitant (p K B 8.72 and 7.54, respectively). In the guinea pig ileum bioassay Pronetupitant antagonized the contractile effect of SP showing a similar potency as Netupitant (p K B ≈ 9). Similar results were obtained with 5 min preincubation time while at 2 min only Pronetupitant produced significant effects. In vivo in mice the intrathecal injection of 0.1 nmol SP elicited the typical scratching, biting and licking (SBL) nociceptive response. This effect of SP was dose dependently (0.1–10 mg/kg) antagonized by Pronetupitant given intravenously 2 h before the peptide. Superimposable results were obtained using Netupitant. Pharmacokinetic studies performed in rats demonstrate that Pronetupitant, after i.v. administration, is quickly (few minutes) and completely converted to Netupitant. Collectively the present results indicated that Pronetupitant acts in vitro as selective NK 1 antagonist more potent than Netupitant. However based on the short half-life measured for Pronetupitant in rats, the in vivo action of Pronetupitant can be entirely interpreted as due to its conversion to Netupitant. [ABSTRACT FROM AUTHOR]

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Background and Purpose: Peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P (SP), neurokinin A (NKA) and B (NKB).Experimental Approach: The following in vitro assays were used: calcium mobilization in cells expressing human recombinant NK receptors, BRET studies of G-protein - NK1 receptor interaction, guinea pig ileum and rat urinary bladder bioassays. Nociceptive behavioural response experiments were performed in mice following intrathecal injection of PWT2-SP.Key Results: In calcium mobilization studies, PWT tachykinin derivatives behaved as full agonists at NK receptors with a selectivity profile similar to that of the natural peptides. NK receptor antagonists display similar potency values when tested against PWT2 derivatives and natural peptides. In BRET and bioassay experiments PWT2-SP mimicked the effects of SP with similar potency, maximal effects and sensitivity to aprepitant. After intrathecal administration in mice, PWT2-SP mimicked the nociceptive effects of SP, but with higher potency and a longer-lasting action. Aprepitant counteracted the effects of PWT2-SP in vivo.Conclusions and Implications: The present study has shown that the PWT technology can be successfully applied to the peptide sequence of tachykinins to generate tetrabranched derivatives characterized with a pharmacological profile similar to the native peptides. In vivo, PWT2-SP displayed higher potency and a marked prolongation of action, compared with SP. [ABSTRACT FROM AUTHOR]

Considering the existence of cross-tolerance between clonidine and morphine besides the same interaction between morphine and WIN 55,212-2 persuaded us to verify this fact between WIN 55,212-2 and clonidine in guinea pig ileum, which is a well-known model to examine the mode of action of cannabinoids and α2-adenoceptor agonists, The rectangular pulses were passed to the 0.5 g stretched ileum segments that were fixed in 20-ml organ bath. PowerLab system and Graphpad Prism were applied to record twitches and analyse the data. Electrically evoked contractions were dose-dependently inhibited by WIN 55,212-2 and clonidine ( p D 2= 8.56 ± 0.41 and 7.65 ± 0.15, respectively)., Tolerance to this effect could be induced by 4-h incubation with WIN 55,212-2 (3 × IC50) ( pD2 = 6.36 ± 0.26, degree of tolerance: 159.32) ( P < 0.01) but not with clonidine (2 × IC50 and 4 × IC50) for different time courses. Dose-response curve for inhibitory action of WIN 55,212-2 was shifted to the right after 4-h incubation with clonidine (3 × 10−10 m) comparing to the untreated tissues ( pD2 = 5.26 ± 0.69, degree of tolerance: 2000) ( P < 0.001). This observation provides the evidence for the cannabinoid-noradrenergic systems interaction in the enteric nervous system as a simplified representative for central nervous system. [ABSTRACT FROM AUTHOR]

Desmostachya bipinnata, Gramineae (Poaceae) family, has been traditionally used in treatment of various disorders like asthma, kidney stone, diarrhoea, wound healing etc. Several extracts ofDesmostachya bipinnatawere prepared.Ex-vivoestimation of contractile responses of guinea pig ileum were measured for prepared extracts against histamine and compared with chlorphenirame. All the extracts were able to contract guinea pig ileum while alcoholic extract was superior among all. Such results indicated that prepared extract possess good anti-histaminic activity, which was further confirmed with histamine induced lethality test. Histamine induced lethality in guinea pigs was prevented when the extracts were administered prior to histamine injection in guinea pigs. Hence, it is concluded that the extracts possess anti-histaminic activity and may possess anti-asthmatic potential. [ABSTRACT FROM AUTHOR]

In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC50 = 7.0 ± 1.9 × 10-5 M), histamine (IC50 = 1.3 ± 0.3 × 10-4 M) and serotonin (IC50 = 8.0 ± 1.4 × 10-5 M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD'2 = 4.48 ± 0.08), shifting the curves to the right with Emax reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC50 = 9.0 ± 0.9 × 10-5 M) and carbachol (EC50 = 4.6 ± 0.7 × 10-5 M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca2+ influx through voltage-gated calcium channels (CaV), since caulerpine slightly inhibited the CaCl2-induced contractions in depolarizing medium without Ca2+, shifting the curves to the right and with Emax reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca2+ influx through CaV. However, other mechanisms are not discarded. [ABSTRACT FROM AUTHOR]

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation ( in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Western-immunoblotting and real time RT-PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOS-expressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS-immunoreactive neurons, protein and mRNA levels were up-regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by Nω-propyl- l-arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R. [ABSTRACT FROM AUTHOR]

Background and Purpose Intrathecally (i.t.) administered nociceptin/orphanin FQ ( N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co-application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide ( NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ μ opioid receptor agonist. Experimental Approach The following in vitro assays were used: calcium mobilization in cells expressing the human NOP or classical opioid receptors and chimeric G proteins, receptor and [35S]-GTPγS binding, [35S]-GTPγS binding in rat spinal cord membranes, guinea pig ileum bioassay. In vivo experiments were performed in monkeys using the tail withdrawal assay. Key Results From calcium mobilization studies [Dmt1]N/OFQ(1-13)-NH2 was selected as the most potent and least selective compound. The mixed NOP/opioid full agonist activity and high affinity of [Dmt1]N/OFQ(1-13)-NH2 was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or [35S]-GTPγS binding studies, at rat spinal cord receptors in [35S]-GTPγS binding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum. In vivo in the tail withdrawal assay in monkeys i.t. [Dmt1]N/OFQ(1-13)-NH2 was able to elicit robust and long-lasting antinociceptive effects. Conclusions and Implications Collectively, these results demonstrate that [Dmt1]N/OFQ(1-13)-NH2 behaves as NOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics. [ABSTRACT FROM AUTHOR]

Abstract: The antispasmodic effect of 4′-methylepigallocatechin (MEC), which was isolated from Maytenus rigida Mart (Celestraceae), was investigated in vitro in guinea pig intestinal segments. In the isolated ileum, MEC (1 nM–100μM) did not modify the ileal spontaneous tonus or the electrically elicited contractions. MEC (8μM) significantly (p <0.01) reduced the submaximal contractions induced by histamine (2μM), carbachol (100μM) and BaCl2 (0.03M). An additive relaxing action (p <0.001) was observed by co-incubation of verapamil (10 nM) and MEC (8μM). Although MEC (1nM–100μM) did not modify the contractions elicited by 60mM KCl, it significantly reduced the CaCl2 contractile response without changing the EC50 (effective concentration of CaCl2 causing 50% of maximum response). In brief, these results show that MEC has a potent ileal spasmolytic effect and blocks spasms induced by specific and nonspecific stimuli. Importantly, the spasmolytic effects were attained at low concentrations and might be related to the symptomatic relief of abdominal pain that is obtained from the use of the M. rigida stem bark. [Copyright &y& Elsevier]

Abstract: The novel NK1 receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK1 cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK B 8.87). In cells expressing NK2 or NK3 receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK B 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1–10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK1 agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID50 1.5mg/kg) or orally (ID50 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK1 antagonist. Thus this molecule can be useful for investigating the NK1 receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK1 receptors has been demonstrated valuable for patients. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Berries of the plant Solanum nigrum Linn (Solanaceae) are used for the treatment of asthma in folk medicine and ancient books. Aim of study: To evaluate potential of the plant berries in the treatment of asthma. Materials and methods: Petroleum ether, ethanol and aqueous extracts of S. nigrum berries (50, 100 and 200mg/kg, i.p.) were screened for the treatment of asthma by the various methods viz. effect on clonidine and haloperidol induced catalepsy, milk-induced leucocytosis and eosinophilia, mast cell stabilizing activity in mice and studies on smooth muscle preparation of guinea pig ileum (in vitro). Active petroleum ether extract was standardized by HPTLC. Results: The petroleum ether extract of S. nigrum berries inhibited clonidine-induced catalepsy significantly but not haloperidol-induced catalepsy. Petroleum ether extract significantly inhibited increased leukocyte and eosinophil count due to milk allergen and showed maximum protection against mast cell degranulation by clonidine. Petroleum ether extract resisted contraction induced by histamine better than other extracts. All the results are dose dependant. Active petroleum ether extract showed presence of antiasthmatic compound, β-sitosterol. Conclusion: The petroleum ether extract of S. nigrum berries can inhibits parameters linked to the asthma disease. [Copyright &y& Elsevier]

Ischemic episodes lead to profound functional and structural alterations of the gastrointestinal tract which may contribute to disorders of intestinal motility. Enhancement of glutamate overflow and the consequent activation of NMDA ( N-methyl--aspartate) receptors may participate to such changes by modulating different enteric neurotransmitter systems, including cholinergic motor pathways. The molecular mechanism/s underlying activation of NMDA receptors in the guinea pig ileum were investigated after glucose/oxygen deprivation ( in vitro ischemia) and during reperfusion. The number of ileal myenteric neurons positive for NR1, the functional subunit of NMDA receptors, and its mRNA levels were unchanged after in vitro ischemia/reperfusion. In these conditions, the protein levels of NR1, and of its phosphorylated form by protein kinase C (PKC), significantly increased in myenteric neurons, whereas, the levels of NR1 phosphorylated by protein kinase A (PKA) did not change, with respect to control values. Spontaneous glutamate overflow increased during in vitro ischemia/reperfusion. In these conditions, the NMDA receptor antagonists, (-)-2-amino-5-phosphonopentanoic acid [()-AP5] (10 μmol L) and 5,7-dichlorokynurenic acid (5,7-diClKyn acid) (10 μmol L) and the PKC antagonist, chelerythrine (1 μmol L), but not the PKA antagonist, H-89 (1 μmol L), were able to significantly depress the increased glutamate efflux. The present data suggest that in the guinea pig ileum during in vitro ischemia/reperfusion, NR1 protein levels increase. Such event may rely upon posttranscriptional events involving NR1 phosphorylation by PKC. Increased NR1 levels may, at least in part, explain the ability of NMDA receptors to modulate a positive feedback on ischemia/reperfusion-induced glutamate overflow. [ABSTRACT FROM AUTHOR]

Abstract: Tolerance to most of the cannabinoid effects has been reported, either in humans or in other species. Pharmacodynamic properties of these agents are perceived to be responsible for this phenomenon. The effect of lithium was examined on tolerance to WIN 55,212-2, a synthetic cannabinoid, in guinea pig ileum, a functional model to examine the mode of action of cannabinoids. Ileum strips were mounted in bath containing 37°C Tyrode, under 0.5g resting tension. The pulses were delivered, via platinum electrode, to the tissue and subsequent twitches were recorded using PowerLab system. WIN 55,212-2 could inhibit electrically stimulated twitches of guinea pig ileum in a dose dependent manner (pD2 =8.56±0.41). Tolerance to this effect could be induced by incubating isolated ileum for 4h with WIN 55,212-2 (3×IC50) (pD2 =6.36±0.26, degree of tolerance: 159.32) (P< 0.01). Lithium (1mM) could interfere with the development of this cannabinoid-induced tolerance and restores the sensitivity of ileum to inhibitory action of WIN 55,212-2 either when strips were exposed to lithium chloride during 4h incubation period (pD2 =7.26±0.39) (P> 0.05 versus non-tolerant group) (degree of tolerance: 20.28), or when lithium was added immediately before examining the inhibitory action of WIN 55,212-2 (pD2 =8.09±0.33) (P< 0.05 versus corresponding tolerant group) (degree of tolerance: 2.96). Upon these results, it appears reasonable to consider an interventional role for lithium which may be able to break the chain of development and/or expression of tolerance to WIN 55,212-2 in this experimental model. [Copyright &y& Elsevier]

Abstract: This study aimed to elucidate the mechanism(s) of the spasmogenic action of Loranthus ferrugineus in isolated guinea pig ileum. Thus the contractile responses of guinea pig ileum to graded additions of either L. ferrugineus methanol extract or its n-butanol fraction were tested in the presence and absence of various pharmacological interventions. The data showed that L. ferrugineus methanol extract and the n-butanol fraction produced a concentration-dependent spasmogenic effect in isolated guinea pig ileum segments. These effects were significantly inhibited in the presence of 1 μM atropine. In contrast, the response to the lowest concentrations of L. ferrugineus methanol extract (0.25, 0.5 and 1 mg/mL) and n-butanol fraction of L. ferrugineus (0.125, 0.25 and 0.5 mg/mL) were considerably enhanced in the presence of 0.05 μM neostigmine. Neither L. ferrugineus methanol extract nor n-butanol fraction contractile responses were affected upon the incubation of the ileal segments with 100 μM hexamethonium. The results of this study show that the spasmogenic effect of L. ferrugineus is possibly mediated through a direct action on intestinal muscarinic receptors. It is suggested that the bioactive constituents of L. ferrugineus serve as a substrate for acetylcholinesterase. [Copyright &y& Elsevier]

In the present study, L. ferrugineus methanol extract (LFME) was evaluated for its blood pressure lowering effect in anesthetized normotensive Sprague Dawley (SD) rats and its spasmogenic effect in isolated guinea pig ileum. The possible mechanism(s) of action were also investigated. LFME was obtained by Soxhlet extraction. The rats were fasted overnight and anesthetized with sodium pentobarbitone (60 mg/kg i.p.). LFME was administered in i.v. boluses in the concentrations of 25, 50, 100 and 200 mg/kg respectively, with concomitant monitoring of mean arterial pressure (MAP). It was found that LFME dose-dependently reduced MAP. An i.v. bolus injection of atropine significantly decreased the blood pressure lowering effect of LFME. Similarly, L-NAME (Nω-nitro-L-arginine methyl ester) significantly lowered both the MAP and the action duration. Conversely, no significant change in MAP was seen following i.v. injections of neostigmine, hexamethonium, prazosin and propranolol. LFME also produced a dose-dependent contractile effect in guinea pig ileum. This contraction was significantly reduced in atropine pre-incubated tissue segments, yet it was significantly enhanced in the presence of neostigmine. No appreciable change in the ability of LFME to contract guinea pig ileum was seen in the presence of hexamethonium. Accordingly, it can be postulated that LFME possesses a marked hypotensive effect that can be attributed to stimulation of muscarinic receptors and/or stimulation of nitric oxide (NO) release. Moreover, LFME retains a considerable spasmogenic action due to its cholinergic properties. The hypotensive and spasmogenic effects of LFME justify its traditional uses. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Mirabilis jalapa Linn is a well-studied plant. The indigenous people of Mexico use Mirabilis jalapa to cure many infirmities including dysentery, diarrhea, muscular pain and abdominal colic. In the present investigation, we have further characterized some pharmacological properties of an extract of Mirabilis jalapa flowers; therefore, we intend to contribute to understand the pharmacological effects and clarify the complex use of this medicinal plant. Results: The extract of Mirabilis jalapa (1–1000μg/mL) exhibits an inhibitory effect (IC50 =18±0.7μg/mL) on gut smooth muscle contractility whereas it stimulates the contraction of rabbit aortic muscle (EC50 =11.60±0.26μg/mL) in a concentration-dependent manner. Conclusions: These effects were not due to either ACh or HIS receptors blockage, IP3, cAMP, cGMP, Ca2+ release from intracellular storage, or protein kinase mediated contraction–relaxation mechanisms. The effects inducted by the Mirabilis jalapa extract may involve a serotoninergic mechanism, which, in turn, interacts with other adrenergic systems. Further studies are necessary to identify the active compounds within the extract and to elucidate the mechanism of action. [Copyright &y& Elsevier]

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca2+-activated K+ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs. [ABSTRACT FROM AUTHOR]

Substance P (SP) is possibly involved in the pathophysiology of depression and anxiety. We investigated interactions between antidepressants on SP-induced effects and their potential calcium-blocking activity in the isolated guinea pig ileum. All the antidepressants tested, except pargyline, moclobemide, mianserin, and reboxetine, were able to inhibit in a concentration-dependent manner the contraction induced by 100 nmol/L SP. Clomipramine, fluoxetine, maprotiline, and amitriptyline (all at 3 μmol/L) flattened the concentration–response curves to SP, resulting in a reduction of up to 59%, 63%, 32%, and 23%, respectively, of the maximum contractile effect. All the antidepressants tested (3 μmol/L), except pargyline, moclobemide, and mianserin, produced a rightward parallel shift of the concentration–response curve to CaCl2. The L-type selective calcium blocker nifedipine and the T-type selective mibefradil showed similar behaviour against both agonists used, SP and CaCl2. The relative order of potency was nifedipine (pA2, 7.6 ± 0.1) > clomipramine (pA2, 7.0 ± 0.1) > fluoxetine (pKB, 6.5 ± 0.1) = mibefradil (pKB, 6.6 ± 0.1) > amitriptyline (pKB, 6.3 ± 0.1) = maprotiline (pKB, 6.2 ± 0.1) > fluvoxamine (pKB, 5.9 ± 0.1). The data reported in the present study suggest that the antidepressants tested did not behave as competitive antagonists versus NK1-receptor subtypes, but their inhibitory action seems to be related to their calcium-blocking properties. [ABSTRACT FROM AUTHOR]

Abstract: The present study describes the antispasmodic activity of some fractions from the bark of Persea cordata, against guinea pig ileum contracted by acetylcholine and histamine. Hexane and dichloromethane fractions demonstrated the most promising biological effects, having similar potency to that of papaverine, a well-known antispasmodic agent. [Copyright &y& Elsevier]

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10−5 m. A dissociation constant ( KB) of 3.6 μm for the antagonist was calculated. At higher concentrations, 3 × 10−5 and 10−4 m, of the antagonist, the agonist dose–response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10−4 m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10−5 m produced two-, three- and four-fold shift of the carbachol dose–response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the α-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine. [ABSTRACT FROM AUTHOR]

Abstract: The involvement of NMDA glutamate receptors in the effects of glucose/oxygen deprivation (in vitro ischaemia) on spontaneous endogenous acetylcholine and glutamate overflow from the guinea pig ileum was studied. Neurotransmitter overflow was measured by HPLC. Deprivation of glucose in the medium slightly reduced acetylcholine overflow, and did not significantly influence glutamate overflow. During oxygen deprivation and glucose/oxygen deprivation, acetylcholine overflow augmented with a biphasic modality: an early peak was followed by a long lasting increase, whereas glutamate overflow increased with a rapid and sustained modality. The effects of glucose/oxygen deprivation on both acetylcholine and glutamate overflow were abolished after reperfusion with normal oxygenated medium. Acetylcholine and glutamate overflow induced by glucose/oxygen deprivation were significantly reduced in the absence of external Ca2+ as well as by the addition of the mitochondrial Na+–Ca2+ exchanger blocker, CGP 37157, and of the endoplasmic reticulum Ca2+/ATPase blocker, thapsigargin. ±AP5, an NMDA receptor antagonist, and 5,7-diCl-kynurenic acid, an antagonist of the glycine site associated to NMDA receptor, markedly depressed glucose/oxygen deprivation-induced acetylcholine and glutamate overflow as well. Our results suggest that in vitro simulated ischaemia evokes acetylcholine and glutamate overflow from the guinea pig ileum, which is partly linked to an increase in intracellular Ca2+ concentration dependent on both Ca2+ influx from the extracellular space and Ca2+ mobilization from the endoplasmic reticulum and mitochondrial stores. During glucose/oxygen deprivation, ionotropic glutamate receptors of the NMDA type exert both a positive feedback modulation of glutamate output and contribute to increased acetylcholine overflow. [Copyright &y& Elsevier]

Abstract: Glutamate was found to be an excitatory neurotransmitter in the enteric nervous system. Although several lines of evidence indicate a role of glutamate in the regulation of gut motility and secretion the physiological significance of glutamatergic transmission is not clear yet. We studied the effect of glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining in longitudinal muscle strips with attached myenteric plexus of guinea pig ileum. l-Glutamate (100μM) significantly enhanced both the evoked [3H]acetylcholine release and the optical density of nicotinamide adenine dinucleotide phosphate-diaphorase positive neurones, i.e. the intensity of staining. The non-competitive N-methyl-d-aspartate receptor antagonist MK-801 (3μM) abolished the stimulatory effect of l-glutamate on acetylcholine efflux. Similarly, the nitric oxide synthase inhibitor N ω-nitro-l-arginine (100μM) significantly reduced the effect of l-glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining. Our data suggest that endogenous nitric oxide seems to mediate the stimulatory effect of glutamate on acetylcholine release from guinea pig myenteric neurons. [Copyright &y& Elsevier]

Aqueous extracts from eight plant species that are popularly used as spasmolytics have been evaluated for these pre-sumed activities. The species included Kalanchoë pinnata (Lam.) Pers. (Crassulaceae), Cymbopogon citratus Stapf. (Gramineae), Gossypium barbadense L. (Malvaceae), Caesalpinia pulcherrima (L.) Schwartz (Caesalpiniaceae), Tagetes erecta L. (Compositae), Bixa orellana L. (Bixaceae), Cassia alata L. (Caesalpiniaceae), and Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae). Potential spasmolytic activity of the extracts was judged by their ability to reduce forces of smooth muscle contraction of a 2-cm-long piece of guinea pig ileum induced by EC 50 acetylcholine (27 ± 5 µg/l) or EC 50 histamine (102 ± 13 µg/l). The dried extracts were used at concentrations of 0.01, 0.1, 1, and 10 mg/ml. Incubations were carried out in Tyrode buffer kept at a temperature of 37°C and mixed with 5% CO 2 in air and were monitored for 30 s with 60-s intervals. Results (means ± SD; n ≥ 3) were expressed relatively to forces of contraction due to EC 50 acetylcholine or EC 50 histamine alone. The extract from K. pinnata reduced the force of contraction due to histamine but not that due to acetylcholine progressively (40% to 95%) with concentrations increasing from 0.01 to 10 mg/ml. At 10 mg/ml, the C. pulcherrima and B. orellana extracts also counteracted only the histamine-induced force of contraction (by about 25% and 50%, respectively). The C. citratus extract decreased the acetylcholine-induced force of contraction by 20% to 60% at 0.1 to 10 mg/ml and that induced by histamine by 60% to 90% at 0.01 to 10 mg/ml. On the other hand, the G. barbadense extract potentiated rather than reduced forces of contraction due to both acetylcholine and histamine (1.2- to 2-fold at 0.01 to 10 mg/ml). The T. erecta extract had such an effect only on the acetylcholine-induced force of contraction (about 2-fold at 10 mg/ml). The use of the former but not the latter sample alone led to an increase in smooth muscle tone that was not reversed by atropine or chlorpheniramine. The C. alata and P. amarus extracts did not significantly modify forces of contraction due to either acetylcholine or histamine. Our results suggest that preparations from K. pinnata , C. citratus , C. pulcherrima , and B. orellana , but not from G. barbadense , T. erecta , C. alata , and P. amarus , may be useful against smooth muscle spasm. These actions were probably mediated by distinct mechanisms. [ABSTRACT FROM AUTHOR]

We have investigated the effect of blockade of Gi/o proteins on the electrical activity of S-myenteric neurons from guinea-pig ileum longitudinal muscle-myenteric plexus preparations. Intracellular recordings were made from either control or pertussis toxin (PTX) treated tissues. PTX is known to disrupt the function of Gi/o proteins. Incubation with PTX (300 ng/ml) induced three main changes in the electrophysiological characteristics of S-neurons: (1) resting membrane potential was more depolarised; (2) fast excitatory postsynaptic potentials (EPSPs) were faster and narrower; and (3) spontaneous fast EPSPs and action potentials were more frequent. Amplitude of slow EPSPs was not modified. This general increase in excitability after blockade of Gi/o proteins shares characteristics with the slow depolarisation induced by long-lasting trains of low-frequency electrical stimulation and could be mediated by similar mechanisms. Electrophysiological activity of S-myenteric neurons could be modulated by an inhibitory tone, which PTX could modify by either pre- or post-synaptic mechanisms, such as inhibition of the tonic release of inhibitory neurotransmitters and/or intracellular signal transduction involving PTX-sensitive Gi/o proteins. [Copyright &y& Elsevier]

The aim of this study was to investigate whether Toxocara canis infection in guinea pigs provokes changes in ileum responsiveness to histamine. Ileum segments from control and T. canis-infected groups were placed at isometric conditions and submitted to various doses of histamine. No changes were observed between controls and T. canis-infected groups at days 3, 6 and 12 after infection. However, at days 18 and 24 after infection, there was a significant increase in ileum responsiveness to histamine in T. canis-infected group. Pre-incubation of ileum segments with 1 mg ml−1 disodium cromoglycate (DSCG) prevented the increased responsiveness to histamine in T. canis-infected guinea pigs and did not affect ileum contractility in non-infected animals. These results indicate that T. canis-infected guinea pigs develop increased intestinal responsiveness to histamine and that DSCG prevents alterations in smooth-muscle contractility. [Copyright &y& Elsevier]

In the present study, the status of α2-adrenoceptors during cholestasis was investigated by the inhibitory effect of clonidine on the electrically stimulated contractions of mice vas deferens (MVD) and guinea pig ileum (GPI). Clonidine inhibited the contractions in both tissues in a dose-dependent manner. Compared to unoperated animals, there was a significant right-shift in the clonidine concentration-curves of both tissues obtained from 5-day bile-duct ligated (BDL) animals (p < 0.01), implying the hyporesponsiveness of α2-adrenoceptors during cholestasis. Chronic treatment with naltrexone (3 mg/kg/day) reversed the right-shift induced by cholestasis in both tissues. Administration of N(ω)-nitro-L-arginine methyl ester (20 mg/kg/day) also partially reversed cholestasis-induced effect on IC50 of clonidine. These two treatments had no effect on IC50 of tissues from controls. Chronic yohimbine treatment (5 mg/kg/day) recovered the effect of cholestasis on MVD, but sensitized the ileum of unoperated and BDL guinea pigs to clonidine to a similar extent, providing evidence for the role of the augmented adrenergic state of cholestasis in the hyporesponsiveness of norepinephrine-releasing neurons of MVD. We concluded that cholestasis is associated with the decreased responsiveness of α2-adrenoceptors and the cholestasis-associated augmented opioidergic tone and increased NO production contribute to this process. [Copyright &y& Elsevier]

Literature data suggest that the inflamed intestine may be subjected to a considerable oxidative stress. Therefore, the aim of the present study was to simulate the oxidative stress in the gastrointestinal tract and to explore its effect on intestinal motility. This was attained by treating isolated segments from the rabbit jejunum and from the guinea pig ileum with 2,2′-Azobis (2-amidinopropane) dihydrochloride (ABAP), which generates peroxyl radicals by thermal decomposition. Treatment of intestinal segments with ABAP reduced the muscarinic cholinergic response to acetylcholine in both preparations and induced a dose-dependent inhibition of the spontaneous contractions in the jejunum, also in the presence of tetrodotoxin. ABAP was found to inhibit the contractile response induced by BaCl2 in guinea pig ileum preparations. This effect was not dose-dependent and it was reversed by Bay-K 8644, which activates voltage operated L-type calcium channels. The rapid and reversible effects of ABAP suggest that it might directly affect L-type calcium channels before lipoperoxidation induction. In conclusion, the results of the present study show that ABAP could be a useful tool to simulate early contractility dysfunctions mediated by oxidative stress. [Copyright &y& Elsevier]

Binding and autoradiographic studies were carried out in guinea pig ileum to examine neurokinin (NK) 1 receptor location and subtypes using the NK-1-selective radioligand [[sup 125] I]Bolton-Hunter[Sar[sup 9] ,Met(O[sub 2] )[sup 11] ]SP. Two membrane preparations were made: (1) longitudinal muscle containing the myenteric plexus and (2) circular muscle containing the interstitial cells of Cajal. In saturation binding studies, the K[sub D] was estimated as 1.3 and 1.0 nmol/l in each preparation, respectively. In competition binding, the rank order of potency was similar in both membrane preparations: SR140333 ≈ CP99994 ≥ [Sar[sup 9] ,Met(O[sub 2] )[sup 11] ]SP ≈ physalaemin ≈ CP96345 (pIC[sub 50] 9.5–8.7) >> neuropeptide γ ≥ septide (pIC[sub 50] 7.8–7.4). Similarly, scyliorhinin I displayed equal affinity in both preparations, although binding was at two sites, of high affinity (pIC[sub 50] 9.1, 30%) and low affinity (pIC[sub 50] 7.2–6.6, 70%). The only competitor to bind differently in the two muscle preparations was scyliorhinin II, which bound to one site with low potency in the circular muscle (pIC[sub 50] 6.9) but to high-affinity (pIC[sub 50] 9.0, 17%) and low-affinity (pIC[sub 50] 6.7, 83%) sites in the longitudinal muscle. In autoradiographic studies, dense specific binding was associated with the myenteric plexus and the inner circular muscle containing the interstitial cells of Cajal, with minimal specific binding to longitudinal and circular smooth muscle. These results suggest that the NK-1 receptor on the interstitial cells and the myenteric plexus is similar. The apparently low numbers of binding sites on intestinal smooth muscle may be due to a low expression of the NK-1 receptor. Alternatively, the radioligand may not recognize the guinea pig ileum muscle NK-1 receptors due to possible minor differences in their sequence or glycosylation.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The leaves of Cassia tora Linn. (Family: Caesalpiniaceae) were soxhlet extracted with methanol. The spasmogenic effects of the extract were evaluated on guinea pig ileum, rabbit jejunum and mice intestinal transit. Antinociceptive activity of the extract was also evaluated in the mice. The LD50 values of the extract in mice were >2000 mg/kg i.p. and p.o. The extract contracted smooth muscles of guinea pig ileum and rabbit jejunum in a concentration-dependent manner. Atropine reversibly blocked this activity. Mepyramine also reduced the contractile amplitude due to the extract in a concentration-dependent manner. The extract increased intestinal transit in mice dose dependently. C. tora extract significantly (P<0.05) reduced the number of acetic acid induced abdominal constrictions in mice and the effect was comparable to that of aspirin (150 mg/kg i.p.). The extract also significantly (P<0.05) reduced the nociceptive response of mice to increased force (g). The effects were dose-dependent. The studies suggest that the use of C. tora, traditionally, as a purgative and in the treatment of other ailments is justifiable. [Copyright &y& Elsevier]

In the present paper, we describe the results obtained from a preliminary pharmacological and biochemical study of the fire coral Millepora complanata, a regular component of coral reefs in the Mexican Caribbean. The protein-containing crude extract obtained from M. complanata (tested from 0.001 to 1000 μg protein/ml) caused a concentration-dependent stimulation of spontaneous contractions of the guinea pig ileum. The extract (EC50=11.55±2.36 μg/ml) was approximately 12-fold less potent than ionomycin (EC50=0.876±0.25 μg/ml) and its maximum induced contraction (1 mg protein/ml) was equivalent to 68% of the response to 60 mM KCl. FPLC size exclusion chromatography of the M. complanta extract afforded 12 primary fractions, of which only FV (containing proteins with molecular weights ranging from 17 to 44 kDa) and FVIII (consisting of peptides with molecular weights lesser than 1.8 kDa) elicited an excitatory effect when tested at the EC50 of the original extract. After incubation in Ca2+-free medium, the ileal response to FV and FVIII was significantly reduced. Blockage of L-type Ca2+ channels with nifedipine (1 μM) inhibited FV and FVIII-evoked contractions. Cd2+ (10 μM), an unspecific blocker of voltage-activated calcium channels, also antagonized FV and FVIII-induced effects, whereas the Na+ channel blocker tetrodotoxin (10 nM) did not significantly affect FV and FVIII responses. These results suggest that the contractions induced by the bioactive fractions obtained from the crude extract of M. complanata are caused mainly by a direct action on smooth muscle cells, via an increase in Ca2+ permeability that occurs, at least partly, through L-type voltage-dependent Ca2+ channels found in the cell membrane of smooth muscle. [Copyright &y& Elsevier]

The pharmacological profile of the non-peptide OP4 receptor (ORL1, LC132) agonist, Ro 64-6198, was investigated, in three electrically stimulated nociceptin/orphanin FQ (NC)-sensitive preparations, namely the mouse and rat vas deferens and the guinea pig ileum. Ro 64-6198 mimicked the inhibitory effect of NC in the three preparations, while showing slow kinetics of action and a slowly reversible effect compared to the fast and immediately and completely reversible effect of the natural peptide. Ro 64-6198 displayed similar pEC50 and Emax values as NC in the mouse and rat vas deferens while it was 100-fold less potent but more efficacious (higher Emax) than NC in the guinea pig ileum. In the rat vas deferens the effects of Ro 64-6198 were antagonised by [Nphe1]NC(1–13)NH2 and J-113397 with pKB values (6.30 and 8.05, respectively) similar to those obtained against NC (6.20 and 7.77, respectively). Naloxone (1 µM) was inactive. In the guinea pig ileum a clear shift of the concentration response curve to Ro 64-6198 was obtained only using a cocktail of antagonists (naloxone + [Nphe1]NC(1–13)NH2 or naloxone + J-113397). In the mouse vas deferens the antagonists were inactive against Ro 64-6198 either when tested alone or in combination. Therefore, Ro 64-6198 behaved as a selective OP4 receptor agonist only in the rat tissue. These results suggest a physiological heterogeneity in OP4 receptors across tissues and species and may explain why, when tested in vivo, Ro 64-6198 mimics the potent anxiolytic effect of NC better in the rat than in the mouse. [ABSTRACT FROM AUTHOR]

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pKi of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 µM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system. [ABSTRACT FROM AUTHOR]

A series of new non-peptide Bradykinin (BK)B-receptor antagonists is reported. These newleads belong to a larger set including bothcommercially or otherwise available potentialcandidates found by proprietary database searchesusing 3D-pharmacophore models as query, and severalbis-benzamidino compounds selected from ourtryptase-like protease inhibitor library on thebasis of topological considerations, derived fromthe same models.Some of these compounds show functional competitiveantagonistic activity, inhibiting {in vitro} the BK-induced contraction of isolated guinea-pig ileum(GPI) and rat uterus with a pA up to 5.3 and7.0, respectively. They display also bindingaffinity (IC up to 0.56 μM) to the BKB-receptor in radioligand binding assays onGPI membrane preparations and on human IMR-90 fetallung fibroblast cells expressing this receptor subtype.Furthermore, the results with the commerciallyavailable compounds, in some cases developed astherapeutics, show that the used 3D-pharmacophoremodel allows to predict to some certainty possibleside actions of potential drugs. [ABSTRACT FROM AUTHOR]

A series of new non-peptide Bradykinin (BK) B-receptor antagonists is reported. These new leads belong to a larger set including both commercially or otherwise available potential candidates found by proprietary database searches using 3D-pharmacophore models as query, and several bis-benzamidino compounds selected from our tryptase-like protease inhibitor library on the basis of topological considerations, derived from the same models. Some of these compounds show functional competitive antagonistic activity, inhibiting in vitro the BK-induced contraction of isolated guinea-pig ileum (GPI) and rat uterus with a pA up to 5.3 and 7.0, respectively. They display also binding affinity (IC up to 0.56 μM) to the BK B-receptor in radioligand binding assays on GPI membrane preparations and on human IMR-90 fetal lung fibroblast cells expressing this receptor subtype. Furthermore, the results with the commercially available compounds, in some cases developed as therapeutics, show that the used 3D-pharmacophore model allows to predict to some certainty possible side actions of potential drugs. [ABSTRACT FROM AUTHOR]