Concentration-dependent Inhibition Of Spontaneous Contractions Research Articles

Diadenosine tetraphosphate activates P2Y1 receptors that cause smooth muscle relaxation in the mouse colon

P2Y1 receptors play an essential role in inhibitory neuromuscular transmission in the gastrointestinal tract. The signalling pathway involves the opening of small conductance calcium activated potassium-channels (Kca2 family) that results in smooth muscle hyperpolarization and relaxation. Inorganic polyphosphates and dinucleotidic polyphosphates are putative neurotransmitters that potentially act on P2Y1 receptors. A pharmacological approach using both orthosteric (MRS2500) and allosteric (BPTU) blockers of the P2Y1 receptor and openers (CyPPA) and blockers (apamin) of Kca2 channels was used to pharmacologically characterise the effect of these neurotransmitters. Organ bath and microelectrodes were used to evaluate the effect of P1,P4-Di (adenosine-5′) tetraphosphate ammonium salt (Ap4A), inorganic polyphosphates (PolyP) and CyPPA on spontaneous contractions and membrane potential of mouse colonic smooth muscle cells. PolyP neither modified contractions nor membrane potential. In contrast, Ap4A caused a concentration-dependent inhibition of spontaneous contractions reaching a maximum effect at 100 μM Ap4A response was antagonised by MRS2500 (1 μM), BPTU (3 μM) and apamin (1 μM). CyPPA (10 μM) inhibited spontaneous contractions and this response was antagonised by apamin but it was not affected by MRS2500 or BPTU. Both CyPPA and Ap4A caused smooth muscle hyperpolarization that was blocked by apamin and MRS2500 respectively. We conclude that Ap4A but not PolyP activates P2Y1 receptors causing smooth muscle hyperpolarization and relaxation. Ap4A signalling causes activation of Kca2 channels through activation of P2Y1 receptors. In contrast, CyPPA acts directly on Kca2 channels. Further studies are needed to evaluate if dinucleotidic polyphosphates are released from inhibitory motor neurons.

Calcitonin Gene-Related Peptide Receptor Blocker Inhibits Spontaneous Activity of Human Ureter

Calcitonin gene-related peptide (CGRP) is present in nerve fibers that innervate the human ureter and may have important influence on the motility of this organ. The aim of our study was to investigate whether CGRP could affect the motility of an isolated human ureter. The tension and intraluminal pressure of the isolated ureteral segments were recorded and registered on a personal computer. Both phasic and tonic contractions of the isolated preparations were measured as area under the tension or pressure recordings. CGRP and CGRP fragment 8-37 were separately added to the organ baths in a cumulative way, thereby gradually increasing their concentration in the baths' solution. Alpha-CGRP did not affect either phasic, spontaneous activity or tone of isolated ureteral segments, as measured by both tension and intraluminal pressure. On the other hand, CGRP 8-37 caused concentration-dependent inhibition of spontaneous contractions of the isolated ureteral segments.

Insight into the possible mechanism of antidiarrheal and antispasmodic activities of piperine

Piperine is a piperidine-ring containing alkaloid and a major constituent of Piper nigrum Linn. and Piper longum Linn. species, belonging to the Piperaceae family. The present study explored their mode of action in gastrointestinal disorders, such as diarrhea and colic. Piperine at the dose of 10 mg/kg provided complete protection from castor oil-induced diarrhea in mice, similar to that of loperamide. In isolated rabbit jejunum preparations, piperine exhibited concentration-dependent inhibition of spontaneous contractions with an EC50 value of 149.1 μM (89.26–249.20, 95% CI). When used to treat high K+ (80 mM)-induced sustained contractions, piperine inhibited such contractions with an EC50 value of 80.86 μM (56.10–116.50, 95% CI), which suggested a calcium channel blocking (CCB) effect. The CCB effect was further confirmed when pretreatment of the tissues with piperine (10–100 μM) caused a rightward shift in the Ca++ concentration–response curves (CRCs) in Ca++-free medium, similar to that caused by verapamil. Loperamide also caused the inhibition of spontaneous and high K+-induced contractions as well as shifted the Ca++ CRCs to the right at concentrations of 1–10 μM. These data indicate that piperine exhibits antidiarrheal and antispasmodic activities, mediated possibly through calcium channel blockade.

Effects of cisplatin and other Pt(II) complexes on spontaneous motility of isolated human oviduct

The toxicity of platinum(II) ion could be significantly modified by coordination with some organic compounds. In our study, the cytotoxicity and the influence of platinum(II) complexes, such as cisplatin, cis-[PtCl(2)(NH(3))(2)], [PtCl(2)(SMC)] and [PtCl(2)(DMSO)(2)] (where SMC is S-methyl-l-cysteine and DMSO is dimethyl sulphoxide) on spontaneous motility of isolated human fallopian tubes were investigated. Cisplatin showed potent pro-apoptotic effects on peripheral blood mononuclear cells (PBMC). However, peripheral blood mononuclear cells were substantially less sensitive to [PtCl(2)(SMC)] and [PtCl(2)(DMSO)], and these compounds showed no toxic effect on PBMC in all concentrations examined. Cisplatin showed concentration-dependent inhibition of spontaneous contractions of the isolated ampulla. (EC(50)=1.14+/-0.03 x 10(-6)M/l, r=0.714, p<0.05) while [PtCl(2)(SMC)] and [PtCl(2)(DMSO)(2)] did not affect spontaneous contractions of isolated fallopian tube ampulla.

Relaxant and antispasmodic effects of extracts of the orchid Encyclia michuacana on isolated guinea pig ileum

The antispasmodic effects of hexane-, chloroform-, methanol- and water-extracts of the orchid Encyclia michuacana were studied in vitro on guinea pig ileum against three spasmogens: acetylcholine (Ach), histamine and barium chloride. The chloroform extract exerted a significant antispasmodic effect on ileum contractions induced by Ach, histamine and barium chloride (IC(50) = 90.64, 73.12 and 115.2 microg/mL, respectively). Furthermore, the chloroform extract of E. michuacana provoked a concentration-dependent inhibition of spontaneous contractions of guinea pig ileum with potencies comparable to those of papaverine. The antagonism against the spasmogens used suggests that the action of the chloroform extract of E. michuacana could be due mainly to the presence of gigantol. Hexane-, methanol- and water-extracts did not elicit any significant spasmolytic activity.

Pharmacological characterization of venoms obtained from Mexican toxoglossate gastropods on isolated guinea pig ileum

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.

Inhibitory effects of selected antiepileptics on spontaneous motility of isolated human oviducts

Spontaneous motility of the human oviduct is an important factor in the regulation of fertility that can be influenced by a variety of substances. In this study we examined the effects of several antiepileptic drugs on spontaneous contractions of isolated human oviducts. The isolated ampullar and isthmic segments of Fallopian tubes, taken from 34 patients with extensive uterine fibroids, were exposed to carbamazepine, lamotrigine, valproic acid, phenobarbital, diazepam and lorazepam. Carbamazepine and lamotrigine produced concentration-dependent inhibition of spontaneous contractions of both ampullar and isthmic isolated preparations, while the other investigated substances did not exhibit any effect. The effective concentrations of carbamazepine and lamotrigine fall within the range of their therapeutic serum concentrations. When choosing antiepileptics for women of reproductive age, the effects of the drug on motility of the Fallopian tubes should also be considered.

Effect of an oxytocin receptor antagonist and rho kinase inhibitor on the [Ca ++] i sensitivity of human myometrium

Objective The purpose of this study was to assess the possible mechanism and effect of oxytocin receptor on the sensitization of the contractile proteins to [Ca ++] i. Study design Myometrial strips were prepared from biopsy specimens that had been taken at term (37–40 weeks of gestation) cesarean delivery from 22 women, before the onset of labor. Simultaneous measurements of [Ca ++] i and tension were performed on spontaneously contracting strips. The effects of a specific oxytocin receptor antagonist, L371,257, and a rho kinase inhibitor, Y-27632, on the [Ca ++] i and tension transients were determined. Results Application of L371,257 (90 nmol/L) to spontaneously contracting muscle strips reduced peak tension to 43%±12% of its original value without affecting peak [Ca ++] i (105%±15%). More tension is developed at each [Ca ++] i during the falling phase, relative to the rising phase of each spontaneous contraction; this asymmetric [Ca ++] i-tension relationship was abolished by L371,257. The subsequent application of oxytocin (3 μmol/L) reversed the effects of the antagonist on tension. An inhibitor of rho kinase (Y-27632) caused a concentration-dependent inhibition of spontaneous contractions without affecting the underlying Ca ++ signals. The addition of oxytocin (1 nmol/L) to spontaneously active myometrium increased both the duration of the Ca ++ signal and the sensitivity of the contractile machinery to [Ca ++] i; only the latter effect was prevented by Y-27632. Conclusion Oxytocin independently regulates the duration of the periodic Ca ++ signals and the sensitivity of the contractile machinery to Ca ++. The latter is likely to be mediated by rho kinase, which is essential for the effective coupling of increases in [Ca ++] i to tension.

Effects of mibefradil on uterine contractility

Mibefradil, a benzimidazolyl tetralol derivative, is a new Ca 2+ channel antagonist which is structurally distinct from other Ca 2+ channel antagonists such as nifedipine, verapamil and diltiazem. It is a very effective antihypertensive agent that is thought to achieve its action via a higher affinity block for low-voltage activated (T) than for high-voltage-activated (L) Ca 2+ channels. Nevertheless, it blocks L-type Ca 2+ channels in several tissues. In the present study, the effects of mibefradil on spontaneous rhythmic contractions and on contractions elicited by CaCl 2 (K +-depolarized preparations) and oxytocin (in low Ca 2+/Ca 2+-free solutions) were investigated on uterus strips from pregnant and non-pregnant rats. Mibefradil (10 −8–3×10 −6 M) caused concentration-dependent inhibition of spontaneous contractions of uterus strips from pregnant and non-pregnant rats with the IC 50 values of 8.83×10 −7 M; 5.94×10 −7 M (amplitude) and 1.03×10 −6 M; 5.48×10 −7 M (frequency), respectively. Mibefradil (3 μM) caused a rightward shift in the concentration–response curves for CaCl 2 in K + (40 mM)-depolarized uterus strips taken from both pregnant and non-pregnant rats. Mibefradil (3 μM) was, however, more potent for antagonising CaCl 2 responses in uterus strips obtained from pregnant rats than in those from non-pregnant rats. Mibefradil (3 μM) had no effect on oxytocin-induced contraction in Ca 2+-free physiological salt solution (PSS) on uterus strips from non-pregnant rats. However, it markedly inhibited oxytocin-induced contraction of pregnant rat uterus strips in Ca 2+-free PSS. Thus, mibefradil probably antagonizes L-type Ca 2+ channels as well as interferes with the intracellular Ca 2+ release mechanism, which would be helpful in the development of a tocolytic agent.

Pharmacological effects of the aqueous extract of Neorautanenia mitis in rodents

The pharmacological effects of the aqueous extract of Neorautanenia mitis (family Papilonaceae) were studied in rodents. Investigations were carried out on acetic acid-induced writhing (pain) in mice and hind paw oedema in rats. The effects of the extract were also studied on the isolated non-pregnant rat uterus and rabbit jejunum. Results showed the extract to possess significant ( P<0.05) dose-dependent anti-nociceptive activity between 12.5 and 50.0 mg/kg p.o. in mice and slight anti-inflammatory activity at 25 and 50 mg/kg p.o. in rats. The extract also produced a concentration-dependent inhibition of the normal rhythmic contraction of the isolated non-pregnant rat uterus. It was found to inhibit oxytocin-induced as well as acetylcholine-induced contractions in the rat uterus. The extract also exhibited concentration-dependent inhibition of spontaneous contractions of the rabbit jejunum. Preliminary phytochemical analysis of the extract revealed the presence of saponin glycosides, flavonoids, tannins and alkaloids. The extract had an intraperitoneal (i.p.) LD 50 of 282.84±3.2 mg/kg in mice. These data corroborate the traditional use of this plant in the treatment of dysmenorrhea.

Characterisation of the atypical beta-adrenoceptor in rabbit isolated jejunum using BRL 37344, cyanopindolol and SR 59230A.

The present study was carried out to further investigate the nature of the beta-adrenoceptor in rabbit jejunum using BRL 37344, a selective beta3-adrenoceptor agonist, cyanopindolol, a beta-adrenoceptor antagonist with blocking activity at beta3-adrenoceptors and SR 59230A, a new selective beta3-adrenoceptor antagonist. Isoprenaline produced a concentration-dependent inhibition of the spontaneous contractions of rabbit jejunum with a pD2 of 7.14. Propranolol (1 microM) shifted the isoprenaline concentration-response curve (CRC) to the right with a concentration-ratio of 5.85, considerably less than would be expected for an action at classical beta-adrenoceptors (estimated pA2 6.66). BRL 37344 also produced a concentration-dependent inhibition of spontaneous contractions with a pD2 of 7.41. The BRL 37344 CRC was unaffected by propranolol (1 microM). In the presence of propranolol (1 microM), cyanopindolol (1 microM) shifted the isoprenaline CRC to the right (concentration-ratio of 21). Cyanopindolol also shifted the BRL 37344 CRC to the right (concentration-ratio of 38). These shifts are consistent with the affinity of cyanopindolol for beta3-adrenoceptors (estimated pA2 values of 7.27 and 7.38 against isoprenaline and BRL 37344, respectively). In the presence of propranolol (1 microM), SR 59230A produced a concentration-dependent rightward shift of the isoprenaline CRC. The Schild plot gave a pA2 value of 7.16, although the slope of the regression line was significantly different from unity (0.65). SR 59230A also produced a concentration-dependent shift of the BRL 37344 CRC. The Schild plot gave a pA2 of 7.58 with the slope of the regression line not significantly different from unity (0.81). The presence of beta3-adrenoceptors mediating relaxation of spontaneous contractions in rabbit jejunum is supported by the relatively poor antagonism of isoprenaline by propranolol, the relaxant effect of BRL 37344 and the antagonism of isoprenaline and BRL 37344 by cyanopindolol and SR 59230A. The lack of simple competitive antagonism of isoprenaline, but not BRL 37344, by SR 59230A may suggest more than one population of atypical beta-adrenoceptor.

Nitric oxide mediated inhibition of contractile activity in the human uterine cervix.

Nitric oxide (NO) is an important modulator of contractile activity in various tissues. The aim of the present study was to investigate the possible existence of an NO system within the human uterine cervix and to study the effects of NO on the cervix in early pregnancy. Cervical tissue specimens were obtained from 24 women in connection with first trimester legal abortion. NADPH diaphorase staining was used to identify nitric oxide synthase activity within the cervical tissue. Cylindrical tissue specimens were mounted in organ bath chambers for isometric registration of contractile activity. The presence of a functional NO system in the cervix was investigated by adding either sodium nitroprusside or spermine NONOate, two different NO donors, or 8-bromo cGMP, an analogue of the second messenger cyclic guanosine monophosphate (cGMP), to the organ baths. Positive NADPH diaphorase staining was clearly observed in the walls of blood vessels, in cervical smooth muscle cells, and cells scattered in the connective tissue. The NO donating drugs sodium nitroprusside and spermine NONOate both caused a dose-dependent inhibition of spontaneous contractile activity with significant inhibition at concentrations of 10(-5) and 10(-7) M respectively. Furthermore, the participation of NO in the regulation of cervical contractility was indicated by a significant concentration-dependent inhibition of spontaneous contractions when 8-bromo cGMP (10(-5)-10(-3) M) was added to the organ baths. The study indicates the existence of an NO system within the human uterine cervix and a role of NO in control of cervical function.

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein.

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle. 2. At 25 degrees C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-CIATP (2.7+/-0.5 microM, n=7) >ATP (12.9+/-1.1 microM, n=9) > or =2-MeSATP (21.9+/-4.8 M, n=4). In the presence of alphabeta-methylene ATP (alphabeta-MeATP, 200 microM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited. 3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP. 4. The A2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1-100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited. 5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to alphabeta-MeATP (200 microM) but were inhibited by ZM 241385 (100 nM). 6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [gamma-32P]-ATP showed that muscular strips rapidly (10-60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57+/-11% (n=3) by 200 microM alphabeta-MeATP. 7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A2A-receptors.

Characterization of the 5-hydroxytryptamine receptor type involved in inhibition of spontaneous activity of human isolated colonic circular muscle.

1. Experiments were carried out to characterize pharmacologically the 5-hydroxytryptamine (5-HT) receptor types which mediate inhibition of spontaneous contractions of the intertaenial circular muscle in human isolated colon. 2. 5-HT caused a reproducible concentration-dependent inhibition of spontaneous contractions of the circular muscle of human colon in vitro with a mean EC50 value of 0.2 microM and 95% confidence limits of 0.1-0.5 microM. No evidence for a contractile action of 5-HT was found. Tetrodotoxin (TTX, 1.5 microM) caused a rightward shift of the concentration-response curve of 5-HT with a concentration-ratio of 2.9. 3. The inhibitory response to 5-HT was mimicked by several indoles with the rank order of potency 5-HT > 5-methoxytryptamine = alpha-methyl-5-HT > 5-carboxamidotryptamine >> 2-methyl-5-HT. 5-Hydroxyindalpine was inactive. 4. The substituted benzamides were agonists with the following rank order of potency, 5-HT > renzapride > zacopride > metoclopramide > cisapride. 5. The inhibitory responses to 5-HT were not inhibited by methysergide (10 microM) or methiothepin (1 microM), which are antagonists selective for 5-HT1-like and 5-HT2 receptors, nor by ondansetron (10 microM) which is an antagonist at 5-HT3 receptors. 6. The inhibitory responses induced by 5-HT and 5-methoxytryptamine were competitively antagonized by a weak 5-HT4 receptor antagonist, tropisetron, with pKB values of approximately 6. Tropisetron had no significant effect on the inhibitory response curve produced by isoprenaline (0.01-100 microM). 7. The pharmacological profile of the 5-HT-evoked relaxations of human colon circular muscle are consistent with activation of a 5-HT4-like receptor.