PGF2α-induced Contractions Research Articles

Calcium signaling cascades differentially regulate PGF2α-induced myometrial contractions in water buffaloes (Bubalus bubalis).

This study unravels the differential involvement of calcium signaling pathway(s) in PGF2α-induced contractions in myometrium of nonpregnant (NP) and pregnant buffaloes. Compared to the myometrium of pregnant animals, myometrium of NP buffaloes was more sensitive to PGF2α as manifested by changes in mean integral tension (MIT) and tonicity. In the presence of nifedipine, myometrial contraction to PGF2α was significantly attenuated in both NP and pregnant uteri; however, mibefradil and NNC 55-0396 produced inhibitory effects only in uterus of pregnant animals, thus suggesting the role of extracellular Ca2+ influx through nifedipine-sensitive L-type Ca2+-channels both in NP and pregnant, but T-type Ca2+ channels seem to play a role only during pregnancy. Entry of extracellular Ca2+ is triggered by enhanced functional involvement of Pyr3-sensitive TRPC3 channels and Rho-kinase pathways as evidenced by a significant rightward shift of the concentration-response curve of PGF2α in the presence of Pyr3 and Y-27632 in NP myometrium. But significant down-expressions of TRPC3 and Rho-A proteins during pregnancy apparently facilitate uterine quiescence. In the presence of Ca2+-free solution and cyclopiazonic acid (SERCA blocker), feeble contraction to PGF2α was observed in both NP and pregnant myometrium which suggests minor role of intracellular source of Ca2+ in mediating PGF2α-induced contractions in these tissues.

Oxytocin Receptor Antagonists, Atosiban and Nolasiban, Inhibit Prostaglandin F2\u03b1-induced Contractions and Inflammatory Responses in Human Myometrium

Oxytocin receptor antagonists (OTR-A) have been developed as tocolytics for the management of preterm labour due to the significant role of oxytocin (OT) in the onset of both term and preterm labour. Similar to OT, prostaglandins (PGs) play key roles in myometrial contractility and cervical ripening. Inhibition of PG synthesis/activity is used to delay preterm birth. Thus, targeting the PG pathway in combination with an OTR-A may be an effective strategy for delaying preterm delivery. In this study, we examined the effects of atosiban and nolasiban on PGF2α-induced contractions and pro-inflammatory responses in human pregnant myometrium. Both OTR-As, atosiban and nolasiban, inhibited PGF2α-induced contractions in a dose-dependent manner (p < 0.001 and p < 0.01, respectively). These inhibitory effects involved the suppression of PGF2α-mediated increase in intracellular calcium levels. In addition, the OTR-As significantly suppressed PGF2α-induced activation of pro-inflammatory pathways such as NF-κB and mitogen activated protein kinases (MAPKs), and the subsequent expression of contraction-associated-protein, COX-2. We have demonstrated that atosiban and nolasiban not only inhibit contractions elicited by OT, but also inhibit contractions and inflammation induced by PGF2α. This suggests a possible crosstalk between OTR and PG receptor signalling and highlights the importance of understanding G protein-coupled receptor interactions/crosstalk in the development of future tocolytics.

Decreased contraction induced by endothelium-derived contracting factor in prolonged treatment of rat renal artery with endoplasmic reticulum stress inducer

Recent evidence suggests that endoplasmic reticulum (ER) stress is involved in the regulation of various physiological functions, including those of the vascular system. However, the relationship between ER stress and vascular function is poorly understood. The endothelial cells control the vascular tone by releasing endothelium-derived relaxing factors and contracting factors (EDCFs). We hypothesized that tunicamycin, an inducer of ER stress, modifies endothelium-dependent contraction and prostaglandins (PGs), a major class of EDCFs, induced contractions in the rat renal artery in rats. An organ-culture technique was used to purely investigate the effects of ER stress on the vascular tissue. We observed that tunicamycin treatment (20μg/mL for 23 ± 1h) did not affect acetylcholine (ACh)-induced relaxation and decreased EDCF-mediated contractions under nitric oxide synthase (NOS) inhibition induced by ACh, ATP, or A23187 (a calcium ionophore) in the renal arteries. Under NOS inhibition, U46619 (a thromboxane A2 mimetic)- and beraprost (a prostacyclin analog)-induced contractions were also decreased in the renal arteries of the tunicamycin-treated group (vs. vehicle), while PGE2- and PGF2α-induced contractions were similar between the groups. Tunicamycin treatment slightly enhanced the contractions induced by phenylephrine, an α1 adrenoceptor ligand. Isotonic high-K+-induced contractions were similar between the vehicle- and tunicamycin-treated groups. Another ER stress inducer, thapsigargin (4μmol/L for 23 ± 1h), also caused substantial reduction of ACh-induced EDCF-mediated contraction (vs. vehicle-treated group). In the cultured renal arteries, tunicamycin and thapsigargin increased the expression of binding immunoglobulin protein (BiP), an ER stress marker. In conclusion, ER stress induction directly affects renal arterial function, especially in reducing EDCF-mediated contractions.

TRPV4 (Transient Receptor Potential Vanilloid 4) Mediates Endothelium-Dependent Contractions in the Aortas of Hypertensive Mice.

The role of TRPV4 (transient receptor potential vanilloid 4) in regulating vascular contraction in hypertensive mice is poorly established. We tested the hypothesis that TRPV4 regulates endothelium-dependent contractions in aortas from hypertensive mice through the activation of cytosolic cPLA2 (phospholipase A2) and COX2 (cyclooxygenase 2) and identified the possible endothelium-derived contracting factor generated by COX2. Using myography, we demonstrated that GSK1016790A (a TRPV4 agonist) and acetylcholine (ACh) trigger endothelium-dependent contractions in aortas from hypertensive mice, and the contractions were abolished with TRPV4 deletion. PLA2 assay and Western blotting showed that cPLA2 activity was higher in salt-induced hypertension and HC067047 or a Ca2+ chelator inhibited cPLA2 activity. Contractions induced by TRPV4 and ACh were inhibited by the cPLA2 inhibitor or removal of extracellular Ca2+ COX2 expression was enhanced in the endothelium from hypertensive mice and contractions induced by TRPV4 or ACh were inhibited by the COX2 inhibitor. Enzyme immunoassay showed that the release of prostaglandin F2α (PGF2α) was increased in hypertensive mice. GSK1016790A or ACh triggered the release of PGF2α and this was inhibited by HC067047, the cPLA2 inhibitor, and COX2 inhibitor. GSK1016790A, ACh, and PGF2α induced contractions were significantly reduced by S18886 in salt-induced hypertensive mice. The present study demonstrates that PGF2α generated by COX2 in the endothelium is the most likely endothelium-derived contracting factor underlying endothelium-dependent, TRPV4-mediated contraction in hypertensive mice. This contraction involved increased intracellular Ca2+ concentrations and cPLA2 activity. These results suggested an important role of TRPV4 in endothelium-dependent contraction in mice during hypertension.

WITHDRAWN: Differential involvement of L- and T-type Ca2+ channels, store-operated calcium channel (TRPC) and Rho-kinase signaling pathway(s) in PGF2α-induced contractions in myometrium of non-pregnant and pregnant buffaloes (Bubalus bubalis)

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Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse.

ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2.

Quercetin, a main flavonoid in onion, inhibits the PGF2α-induced uterine contraction in vitro and in vivo

Dysmenorrhoea is considered to be caused by excessive levels of prostaglandins, which stimulate abnormal uterine contractions. This study aimed to investigate the modulatory effects of quercetin that is a main flavonoid in onion on uterine contractility and its possible underlying mechanisms. In vitro and in vivo contractile activities of the uteri were determined using uterine horns isolated from adult rats. The results indicated that (1) for contractions induced by PGF2α-, oxytocin-, and carbachol, quercetin showed the most potent suppressing effect among the tested flavonoids; (2) Ca2+-dependent uterine contractions were inhibited by quercetin; (3) quercetin reduced the PGF2α-elicited Ca2+ responses in human uterine smooth muscle cells; (4) quercetin inhibited uterine contractions stimulated by Ca2+ channel activator and depolarisation in response to high K+; (5) quercetin was able to block Ca2+ influx through voltage-operated Ca2+ channels in plasma membrane; and (6) quercetin effectively reduced PGF2α-induced contractions through the administration of increasing doses of 10, 25, and 50 mg/kg in rats. The present findings suggest that onion's major active compound, quercetin, may be a potential adjuvant for treating uterine disorders.

A first-in-human study of PDC31 (prostaglandin F2 receptor inhibitor) in primary dysmenorrhea

What is the safe and pharmacodynamically active dose range for PDC31 (prostaglandin F2α receptor inhibitor) in patients with primary dysmenorrhea (PD)? The 1 mg/kg/h dose of PDC31 appears to be safe and potentially effective in reducing intrauterine pressure (IUP) and pain associated with excessive uterine contractility when given as a 3-h infusion in patients with PD. PDC31 has previously been shown to reduce the duration and strength of PGF2α-induced contractions in human uterine myometrial strip models and to delay delivery in animal models of preterm labor. This was a prospective, multi-center, dose-escalating first-in-human Phase I study conducted from March 2011 to June 2012. A total of 24 women with PD were enrolled and treated with one of five doses (0.01, 0.05, 0.15, 0.3, 0.5 and 1 mg/kg/h) of PDC31 given as a 3-h infusion. Patients were observed for a further 24 h. This study was conducted at four hospitals in Europe in non-pregnant, menstruating women with PD. Women with PD (n = 24) received PDC31 infused over 3 h within 8-10 h of the onset of menstruation. IUP and pain monitoring through the visual analog scale (VAS) was assessed prior to, during and following the infusion. Patients were observed for dose-limiting toxicities and other adverse events. Pharmacokinetic samples were also taken to profile the drug. A 3-h infusion of PDC31 was safe up to and including doses of 1 mg/kg/h. Most adverse events were mild (n = 15; 83.3%) and not considered associated with PDC31 (n = 14; 77.8%). PDC31 infusion decreased uterine activity based on IUP and pain (VAS) scores. IUP was decreased by 23% over all dose levels, reaching a minimum at 135-150 min. There appeared to be a dose-dependent effect on IUP, with the high dose group (1 mg/kg/h) showing the largest decrease in IUP. There was a statistically significant linear dose-effect and concentration-effect relationship for several IUP parameters over the evaluation period of 60-180 min. A dose differentiating effect on pain was seen with the two highest doses. PDC31 demonstrated uncomplicated, linear pharmacokinetics with a terminal half-life of ∼2 h. This was a first-in-human study and exposure to PDC31 was limited for safety reasons. As such, pharmacodynamic parameters were assessed at a two-sided Type I error of 20%, an appropriate level for the exploratory nature of this study without a placebo control arm. This limited the chance of false positive findings to one in five. Like PD, preterm labor is associated with prostaglandin-mediated uterine contractions; therefore, the findings of this study support further development of PDC31 as a treatment for both PD and preterm labor. This work was funded by PDC Biotech GmbH, Vienna, Austria. B.B., R.M.L., L.W., R.J.S., K.J.B. and C.F.S. received reimbursement for the conduct of this study from PDC Biotech GmbH. W.H., M.S. and R.P.S. are paid consultants for PDC Biotech GmbH. P.G. is a paid consultant and shareholder of PDC Biotech GmbH. NCT01250587 at www.clinicaltrials.gov.

Two-step inhibitory effect of kanzo on oxytocin-induced and prostaglandin F2α-induced uterine myometrial contractions.

We previously reported that shakuyaku-kanzo-to, a kampo medicine consisting of shakuyaku and kanzo, has an inhibitory effect on myometrial contractions in pregnant women. In this study, we evaluated the effects of kanzo, glycyrrhizin (a major component of kanzo), glycyrrhetinic acid (GA; a major metabolite of glycyrrhizin), shakuyaku, and paeoniflorin (a major component of shakuyaku) on agonist-induced contractions of the uterus of pregnant humans and rats. We prepared myometrial strips from the uterus of pregnant humans and rats and induced contractions with oxytocin (50 μU/mL) or prostaglandin F2α (PGF2α) (10(-7) or 10(-6)M). Kanzo (250μg/mL) and GA (5×10(-6)M) inhibited the oxytocin-induced and PGF2α-induced contractions in pregnant human and rat myometrium, but shakuyaku (250μg/mL), paeoniflorin (10(-5)M), and glycyrrhizin (10(-5)M) did not inhibit contractions in either. Interestingly, kanzo and GA showed an inhibitory effect after temporarily enhancing the PGF2α-induced contractions in the rat myometrium, but not in the human myometrium. These results suggest that kanzo has at least a two-step inhibitory effect on the myometrial contractions that originate from the kanzo itself and a metabolite of glycyrrhizin in kanzo. Furthermore, kanzo was found to be safe for inhibiting PGF2α-induced contractions in humans because it did not temporarily enhance PGF2α-induced contractions.

Inhibition of activated ERK1/2 and JNKs improves vascular function in mouse aortae in the absence of nitric oxide

Activation of mitogen-activated protein kinases (MAPKs) is important for vascular contraction. Decreased nitric oxide availability combined with activation of MAPKs contributes to an increase in vascular tone. In this study, we have determined the involvement of extracellular signal-regulated kinases1/2 (ERK1/2) and c-Jun N-terminal kinases (JNKs) in reactivity of mouse aortae in the absence of nitric oxide. Additionally, we have examined the contribution of these kinases to endothelium-dependent and prostaglandin F 2α (PGF 2α)-induced contractions. Precontracted aortic rings were treated with MAPK/ERK kinase1/2 (MEK1/2) inhibitor U0126 or JNKs inhibitor SP600125 to determine reactivity after inhibition of nitric oxide synthase using organ bath chambers. Additionally, rings were pretreated with or without these inhibitors to assess PGF 2α- and acetylcholine-induced, endothelium-dependent contractions. Specificity of the inhibitors was evaluated in each aortic ring by determining the phosphorylation levels of ERK1/2 and c-Jun using Bio-Plex™ phospho-protein detection kit. In the absence of nitric oxide both inhibitors caused relaxation, and the dilator response was increased by 2.5-fold using SP600125 in comparison with U0126. Transient endothelium-dependent contractions were blocked by U0126, whereas SP600125 strongly attenuated sustained PGF 2α-induced contractions. U0126 inhibited only phosphorylation of ERK1/2, while SP600125 at higher concentrations not only inhibited phosphorylation of c-Jun but also ERK1/2 phosphorylation. In conclusion, the present study demonstrates that in aortae inhibition of activated ERK1/2 and JNKs mediates vascular relaxation, even in the absence of nitric oxide. Activation of ERK1/2 contributes predominantly to transient endothelium-dependent contractions while JNKs, possibly synergistically with ERK1/2, leads to sustained PGF 2α-induced contractions.

Effects of progesterone on motility and prostaglandin levels in the distal guinea pig colon

Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating Galpha(q/11) proteins that mediate contraction, by upregulating Galpha(s) proteins that mediate relaxation, and by altering the pattern of cyclooxygenase (COX) enzymes and prostaglandins. We aimed to examine whether P4 treatment of guinea pigs in vivo affects basal colon motility [basal motility index (MI)] by altering the levels and actions of PGF(2alpha) and PGE(2). Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the PGF(2alpha)-induced contraction, and PGE(2)-induced inhibition of contraction were examined in muscle strips and cells. The levels of PGF(2alpha) and PGE(2) were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of PGF(2alpha), and PGF(2alpha)-induced contraction. P4 increased PGE(2) levels, and PGE(2) induced relaxation. Pretreatment with IM RU-486 (10 mg/kg per day), a P4 receptor antagonist, 1 h before P4 blocked the actions of P4. The PGF(2alpha) antagonist Al-1180 abolished basal MI and PGF(2alpha)-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on PGF(2alpha) and PGE(2) effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased PGF(2alpha) levels; GTPgammaS increased and GDPbetaS decreased the levels of PGF(2alpha). Galpha(q/11) protein antibodies (Abs) reduced PGF(2alpha) levels, and Galpha(i3) Abs blocked its motor actions. Galphas Abs increased PGF(2alpha) but decreased PGE(2) levels. We concluded that P4 decreases basal MI by reducing PGF(2alpha) levels caused by downregulation of Galpha(q/11) and that PGF(2alpha)-induced contraction was blocked by downregulating Galpha(i3). P4 also decreased the basal MI by increasing PGE(2) levels, and PGE(2) induced relaxation by upregulating Galpha(s) proteins.

Cyclooxygenase-2–Derived Prostaglandin F 2α Mediates Endothelium-Dependent Contractions in the Aortae of Hamsters With Increased Impact During Aging

Hypertension and vascular dysfunction result in the increased release of endothelium-derived contracting factors (EDCFs), whose identity is poorly defined. We tested the hypothesis that endothelial cyclooxygenase (COX)-2 can generate EDCFs and identified the possible EDCF candidate. Changes in isometric tension of aortae of young and aged hamsters were recorded on myograph. Real-time changes in intracellular calcium concentrations ([Ca(2+)](i)) in native aortic endothelial cells were measured by imaging. Endothelium-dependent contractions were triggered by acetylcholine (ACh) after inhibition of nitric oxide production and they were abolished by COX-2 but not COX-1 inhibitors or by thromboxane-prostanoid receptor antagonists. 2-Aminoethoxydiphenyl borate (cation channel blocker) eliminated endothelium-dependent contractions and ACh-stimulated rises in endothelial cell [Ca(2+)](i). RT-PCR and Western blotting showed COX-2 expression mainly in the endothelium. Enzyme immunoassay and high-performance liquid chromatography-coupled mass spectrometry showed release of prostaglandin (PG)F(2alpha) and prostacyclin (PGI(2)) increased by ACh; only PGF(2alpha) caused contraction at relevant concentrations. COX-2 expression, ACh-stimulated contractions, and vascular sensitivity to PGF(2alpha) were augmented in aortae from aged hamsters. Human renal arteries also showed thromboxane-prostanoid receptor-mediated ACh- or PGF(2alpha)-induced contractions and COX-2-dependent release of PGF(2alpha). The present study demonstrates that PGF(2alpha), derived from COX-2, which is localized primarily in the endothelium, is the most likely EDCF underlying endothelium-dependent, thromboxane-prostanoid receptor-mediated contractions to ACh in hamster aortae. These contractions involved increases in endothelial cell [Ca(2+)](i). The results support a critical role of COX-2 in endothelium-dependent contractions in this species with an increased importance during aging and, possibly, a similar relevance in humans.

Uterine relaxant effects of Curcuma aeruginosa Roxb. rhizome extracts

The effects and plausible mechanism of action of Curcuma aeruginosa Roxb. (Zingiberaceae) rhizome chloroform and methanol extracts on the uterine contraction were investigated using isolated uterus strips from estrogen primed rats . The contractile responses were recorded isometrically with a Grass FT03 force transducer connected to a MacLab system. The experiments were carried out on both nonstimulated, agonist- and KCl-stimulated uteri. In the nonstimulated uterus, the two extracts (10–400 μg/ml) had no significant effect. In contrast, in the stimulated uterus, the chloroform and methanol extracts exerted concentration-dependent inhibition of the contractions induced by oxytocin (1 mU/ml), prostaglandin F 2α (PGF 2α, 0.5 μg/ml), ACh (3 × 10 −6 M) and KCl (40 mM) with the IC 50 (inhibition of force) of 31.4, 58.59, 56.21 and 29.28 μg/ml; and 57.79, 69.3, 223.8 and 69.19 μg/ml, respectively. Verapamil, the reference L-type calcium channel blocker, exhibited a similar pattern of inhibition with the IC 50 of 0.03, 0.25, 0.35 and 0.04 μg/ml. The IC 50 of diclofenac against a PGF 2α-induced contraction was 31.36 μg/ml. It is known that the contraction induced by agonists and KCl is mainly due to calcium influx through the voltage-gated L-type calcium channels opened indirectly or directly by agonist–receptor activation and KCl. Thus, it is speculated that the two plant extracts might inhibit uterine contraction by interrupting the influx of Ca 2+ probably through voltage-gated L-type calcium channels. This possibility was further substantiated by the ability of the extracts to shift the CaCl 2–contraction curves to the right. As the methanol extract also reduced the contraction of oxytocin in Ca 2+-free EDTA solution; thus, it is suggested that part of its action may be involved with an intracellular mechanism. The effect of the two extracts did not involve the activation of β 2-adrenoceptors since their effects were unaffected by propranolol. Based on the inhibitory effect of the extracts on the oxytocin-induced contraction, it is concluded that the extracts might be useful as tocolytic agents for the prevention of preterm labor. Their effects on the inhibition of PGF 2α-induced contractions also seem useful for the treatment of dysmenorrhea. There are reports by others that the plant rhizome contains β-pinene and sesquiterpenes. In addition, there is evidence that these compounds possess spasmolytic effects in the rat intestine and uterus. Therefore, the uterine relaxant effect of the plant extracts could be due to β-pinene and some sesquiterpene lactones contents. The methanol extract is less potent than the chloroform extract, and this might be due to the lower amount of terpene compounds or different compounds may involve in this action.

17β-Estradiol inhibits calcium-dependent and -independent contractions in isolated human saphenous vein

Studies suggest that estrogen modulate vascular reactivity but at present its exact mechanism of action has yet to be clarified. The aim of this study was to evaluate the effect of 17β-estradiol (E2) on calcium-dependent and -independent contractions induced in the human saphenous veins (HSVs). HSVs were obtained from patients undergoing coronary artery bypass graft surgery. The ability of E2 to modulate Ca 2+ entry was assessed by obtaining concentration–response curve to CaCl 2 in the absence or presence of E2. In other experiments intracellular Ca 2+ was depleted by repeated application of phenylephrine in the presence of cyclopiazonic acid (CPA). Then, at the plateau of PGF 2α contraction, E2 or nifedipine (NIF) was added. Involvement of protein kinase C (PKC) in relaxant effect of E2 was evaluated by application of phorbol-12,13-dibutyrate (PDBu) in normal or Ca 2+-free Krebs’ solution. When the contraction was obtained, E2 or NIF was added. In Ca 2+-free hyperpolarizing solution, pretreatment with E2, concentration dependently reduced contractions induced by cumulative addition of calcium chloride. Furthermore, E2 elicited relaxant effects on the PGF 2α-induced contractions in Ca 2+-free solution in the presence or absence of CPA. Both E2 and NIF produced significant relaxation in HSV rings contracted by direct activation of PKC in Krebs’ solution. However, in Ca 2+-free solution, NIF failed to induce relaxant effect but E2 kept its effect on the PDBu-induced contraction. These results suggest that the relaxant effect of E2 on HSV is elicited by calcium-dependent and -independent pathways. The calcium-independent pathway may involve PKC inhibition.

H-ras Inhibits RhoA/ROCK Leading to a Decrease in the Basal Tone in the Internal Anal Sphincter

The present studies evaluated the role of H-ras and its implications in the RhoA/Rho kinase (ROCK) pathway in regulating basal tone in the internal anal sphincter (IAS). Studies were performed in the IAS from the wild-type (H-ras(+/+)) and knock-out (H-ras(-/-)) mice. The basal tone of smooth muscle strips was measured by isometric force transducers. Length of smooth muscle cells (SMC) isolated from the IAS in the basal state was determined by phase contrast microscopy. Experiments were repeated in the presence of Y 27632, a ROCK inhibitor. Involvement of the RhoA/ROCK machinery was analyzed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Reversal of H-ras knock-out effect was evaluated by transfection of SMCs with the constitutively activated (G12V) mutant. Basal tone of the H-ras(-/-) IAS was significantly higher and resistant to relaxation by Y 27632, compared with the H-ras(+/+) IAS. Similarly, the length of SMCs from H-ras(-/-) IAS was significantly shorter. Y 27632 eliminated this difference. RhoA immunoreactivity shifted from cytoplasm to plasma membrane in H-ras(-/-) SMCs, a change typically associated with contraction. Further, SMCs from H-ras(-/-) mice exhibited higher levels of the contractile proteins ROCK II, phosphorylated-MYPT(1) and phosphorylated-MLC(20). Transfection with the G12V mutant increased the length of H-ras(-/-) cells. Conversely, the dominant negative H-ras (S17N) mutant decreased the length of H-ras(+/+) cells. H-ras negatively regulates basal tone in the IAS by inhibiting RhoA/Rho-kinase machinery. Studies may have significant relevance in the pathophysiology and therapy of certain anorectal motility disorders associated with the IAS dysfunction.

The complex venous response to the ETB receptor (ETBR) agonist sarafotoxin S6C

Arteries and veins are both actively engaged in the control of cardiovascular function. However, they display many functional differences, such as the involvement in the endothelin system. In the endothelium, ETBR mediates nitric oxide (NO) release and relaxation, while on smooth muscle cells (SMCs) it mediates contraction. Although the ETBR is present on SMCs of both rat aorta (RA) and vena cava (RVC), stimulation with S6C, a selective ETBR agonist, leads to contraction of the RVC, but not RA. We hypothesized that the venous endothelial ETBR is less functionally active than the arterial one. Therefore we studied the ability of S6C to act as a relaxing agent when used on contracted, endothelium-intact RA and RVC. In PGF2α (20 μM)-contracted RA, S6C (100 nM) induced a relaxation (21.5±4.8% PGF2α-induced contraction). By contrast, in the RVC, S6C induced a four-phase response (short relaxation-short contraction-long relaxation-long contraction). BQ788, the ETBR antagonist (1 μM), blocked the S6C-induced relaxation in the aorta, while it only altered the short contractile phase of the VC response. The RVC response to S6C cannot be explained by a different ability of venous and arterial endothelium to produce NO, since ACh (10 μM) induced a similar relaxation of RA and RVC, which was completely blocked by LNNA (100 μM). These data highlight increasingly complex functions of the ETBR in the cardiovascular system.

The Effects of Ginger Oils on Rat Uterine Contraction

Ginger, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), has been extensively studied for its pharmacological activities [1]. It produced inhibitory effects on vascular and gut contractility [2, 3]. However, its effects on uterine contractility have not been elucidated. The aims of the study were to investigate the effects of ginger on rat uterine contraction. We examined the effects of ginger oils on phasic contractions arising either spontaneously or PGF2α stimulation and the mode of action. Ginger oils were obtained by water distillation. Rats were killed by asphyxiation with CO2 and longitudinal uterine smooth muscles isolated. Isometric force was measured and the effects of ginger oils studied. The results showed that at each concentration (10–100 microL/100mL) ginger oils reduced spontaneous contractions, and that the effect was dose dependent. The PGF2α-induced contractions were significantly reduced by ginger oils. Increases in external calcium concentration completely reversed the relaxant effects of ginger oils. In conclusion, ginger oils are potent inhibitors of phasic activity in rat uterus, irrespective of how it is produced, and our data suggest their effects lie at the surface membrane.

Characterisation of the relaxant response to raloxifene in porcine coronary arteries

The present study characterises the vasorelaxant response to raloxifene in isolated rings of porcine coronary artery. Tissues precontracted either with KCl (30 mM) or prostaglandin F 2α (PGF 2α; 3 μM) were concentration-dependently relaxed by raloxifene (0.1–10 μM). Relaxation was not inhibited by the estrogen receptor antagonist 7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17 β-diol (ICI 182,780; 1 μM). Preincubation with raloxifene (1–3 μM) caused an inhibition of the KCl or PGF 2α-induced contraction. The effects of raloxifene were independent of the endothelium. The relaxant response to raloxifene was slow in the onset and could not be reversed after repeated washings. Raloxifene did not affect Ca 2+ release from intracellular stores since it failed to inhibit a transient contraction induced by caffeine (10 mM). Raloxifene-induced relaxation was not influenced by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM; 10–20 μM). Calcium-induced contractions in Ca 2+-free high K + (60 mM) depolarising medium were concentration-dependently inhibited by raloxifene (0.3–3 μM). If arterial rings were incubated with the L-type Ca 2+ channel activator ( S)-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (( S)-(–)-Bay K 8644; 0.1 μM), cumulative concentration–response curves to Ca 2+ were shifted to the left. Raloxifene (0.3–3 μM) inhibited the effect of ( S)-(–)-Bay K 8644 in a concentration-dependent manner. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1 H-imidazole (SB 203580; 10 μM), an inhibitor of p38 mitogen-activated protein kinase (MAPK), diminished raloxifene-induced relaxation in endothelium-denuded arterial rings. Western blot analysis demonstrated that raloxifene stimulated p38 MAPK. It is concluded that raloxifene has an inhibitory effect on voltage-gated and receptor-operated L-type Ca 2+ channels in porcine coronary arteries, thus inducing vascular relaxation independent of the endothelium. p38 MAPK is, at least in part, involved in the relaxant response to raloxifene.

Ligustilide inhibits spontaneous and agonists- or K + depolarization-induced contraction of rat uterus

In the present study, the effects of ligustilide (LIG) on uterine contraction in vitro were investigated. In isolated rat uterine, LIG (2–8 μg/ml) inhibited the spontaneous periodic contraction in a concentration-dependent manner (EC 50 = 4.4 μg/ml, 95% confidence interval 2.7–6.1 μg/ml), and attenuated prostaglandin F2alpha (PGF 2α)- or acetylcholine chloride (Ach)-induced uterine contractions. At 8 μg/ml, LIG nearly completely blocked the PGF 2α-induced contractions (95.3%). In the case of Ach-induced contraction, about 73.9% was inhibited by LIG at this dosage. It was also observed that LIG affected significantly oxytocin-induced increase in the contraction of uterine horns that were incubated not only in the Locke solution but also in a Ca 2+-free solution. In addition, LIG caused concentration-dependent inhibition of uterine contraction induced by K + (56.3 Mm) depolarization, reaching the significant level at 2 μg/ml (EC 50 = 3.3 μg/ml, 95% confidence interval 2.5–4.1 μg/ml). The findings clearly show that LIG has multiple effects on the uterine smooth muscles, suggesting that LIG possesses a non-specific antispasmodic function. The data also imply strongly that LIG is one of active ingredients of Danggui and has the potential to be developed into an effective drug for the prevention and treatment of primary dysmenorrhoea.

Vasorelaxant effects of glibenclamide on rat thoracic aorta: sıçan torasik aortasında glibenklamidin vazorelaksan etkileri

The purpose of this study was to investigate the effect of glibenclamide, a selective ATP-sensitive K+(KATP) channel blocker, on agonist-induced contractions in isolated rat aortic rings. The possible involvement of endothelium in glibenclamide-induced response was also investigated. The concentrationresponse curves of prostaglandin F2α (PGF2α; 0.1 nM-30 µM), serotonin (5-hydroxytryptamine, 5-HT; 1 nM30 µM) and phenylephrine (1 nM-30 µM) were obtained in the absence or presence of glibenclamide (1, 3 or 10 µM). Maximum agonist-induced contractions in the presence of glibenclamide 1, 3 and 10 µM were decreased by 30±5, 64±3 and 86±2 percent for PGF2αand 41±11, 50±11 and 65±5 percent for 5-HT respectively. Phenylephrine-induced contractions remained unaltered by glibenclamide 10 µM. The inhibitory effect of glibenclamide (10 µM) on PGF2α- or 5-HT-induced contractions were attenuated upon Nω-nitro-L-arginin (L-NNA; 100 µM) plus indomethacin (10 µM) incubation. The inhibitory effect of glibenclamide on PGF2α-induced contractions was also assessed in endothelium-denuded arteries and the inhibitory effect of glibenclamide was greater in endothelium-denuded arteries than in endothelium-intact arteries pretreated with L-NNA plus indomethacin (64±4% versus 46±10%, respectively). These results suggest that glibenclamide could act through multiple sites in either endothelium or the underlying arterial smooth muscle and thus serve as a non-selective muscle relaxant at high concentration