Biphasic Contraction Research Articles

Regulation of nerve-evoked contractions of the murine vas deferens

Stimulation of sympathetic nerves in the vas deferens yields biphasic contractions consisting of a rapid transient component resulting from activation of P2X1 receptors by ATP and a secondary sustained component mediated by activation of α1-adrenoceptors by noradrenaline. Noradrenaline can also potentiate the ATP-dependent contractions of the vas deferens, but the mechanisms underlying this effect are unclear. The purpose of the present study was to investigate the mechanisms underlying potentiation of transient contractions of the vas deferens induced by activation of α1-adrenoceptors. Contractions of the mouse vas deferens were induced by electric field stimulation (EFS). Delivery of brief (1s duration) pulses (4 Hz) yielded transient contractions that were inhibited tetrodotoxin (100 nM) and guanethidine (10 µM). α,β-meATP (10 µM), a P2X1R desensitising agent, reduced the amplitude of these responses by 65% and prazosin (100 nM), an α1-adrenoceptor antagonist, decreased mean contraction amplitude by 69%. Stimulation of α1-adrenoceptors with phenylephrine (3 µM) enhanced EFS and ATP-induced contractions and these effects were mimicked by the phorbol ester PDBu (1 µM), which activates PKC. The PKC inhibitor GF109203X (1 µM) prevented the stimulatory effects of PDBu on ATP-induced contractions of the vas deferens but only reduced the stimulatory effects of phenylephrine by 40%. PDBu increased the amplitude of ATP-induced currents recorded from freshly isolated vas deferens myocytes and HEK-293 cells expressing human P2X1Rs by 93%. This study indicates that: (1) potentiation of ATP-evoked contractions of the mouse vas deferens by α1-adrenoceptor activation were not fully blocked by the PKC inhibitor GF109203X and (2) that the stimulatory effect of PKC on ATP-induced contractions of the vas deferens is associated with enhanced P2X1R currents in vas deferens myocytes.

Pharmacological characterisation of the CB1 receptor antagonist activity of cannabidiol in the rat vas deferens bioassay

Cannabidiol is increasingly considered for treatment of a wide range of medical conditions. Binding studies suggest that cannabidiol binds to CB1 receptors. In the rat isolated vas deferens bioassay, a single electrical pulse causes a biphasic contraction from nerve-released ATP and noradrenaline. WIN 55,212-2 acts on prejunctional CB1 receptors to inhibit release of these transmitters. In this bioassay, we tested whether cannabidiol and SR141716 were acting as competitive antagonists of this receptor. Monophasic contractions mediated by ATP or noradrenaline in the presence of prazosin or NF449 (P2X1 inhibitor), respectively, were measured to a single electrical pulse delivered every 30 min. Following treatment with cannabidiol (10–100 μM) or SR141716 (0.003–10 μM), cumulative concentrations of WIN 55,212–2 (0.001–30 μM) were applied followed by a single electrical pulse. The WIN 55,212–2 concentration-contraction curve EC50 values were applied to global regression analysis to determine the pKB. The antagonist potency of cannabidiol at the CB1 receptor in the rat vas deferens bioassay matched the reported receptor binding affinity. Cannabidiol was a competitive antagonist of WIN 55,212–2 with pKB values of 5.90 when ATP was the effector transmitter and 5.29 when it was noradrenaline. Similarly, SR141716 was a competitive antagonist with pKB values of 8.39 for ATP and 7.67 for noradrenaline as the active transmitter. Cannabidiol's low micromolar CB1 antagonist pKB values suggest that at clinical blood levels (1–3 μM) it may act as a CB1 antagonist at prejunctional neuronal sites with more potency when ATP is the effector than for noradrenaline.

Cytochrome P450 epoxygenase-derived 5,6-epoxyeicosatrienoic acid relaxes pulmonary arteries in normoxia but promotes sustained pulmonary vasoconstriction in hypoxia.

The aim of the study was to investigate the role of cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in sustained hypoxic pulmonary vasoconstriction (HPV). Vasomotor responses of isolated mouse intrapulmonary arteries (IPAs) were assessed using wire myography. Key findings were verified by haemodynamic measurements in isolated perfused and ventilated mouse lungs. Pharmacological inhibition of EET synthesis with MS-PPOH, application of the EET antagonist 14,15-EEZE or deficiency of CYP2J isoforms suppressed sustained HPV. In contrast, knockdown of EET-degrading soluble epoxide hydrolase or its inhibition with TPPU augmented sustained HPV almost twofold. All EET regioisomers elicited relaxation in IPAs pre-contracted with thromboxane mimetic U46619. However, in the presence of KCl-induced depolarization, 5,6-EET caused biphasic contraction in IPAs and elevation of pulmonary vascular tone in isolated lungs, whereas other regioisomers had no effect. In patch-clamp experiments, hypoxia elicited depolarization in pulmonary artery smooth muscle cells (PASMCs), and 5,6-EET evoked inward whole cell currents in PASMCs depolarized to the hypoxic level, but not at their resting membrane potential. The EET pathway substantially contributes to sustained HPV in mouse pulmonary arteries. 5,6-EET specifically appears to be involved in HPV, as it is the only EET regioisomer able to elicit not only relaxation, but also sustained contraction in these vessels. 5,6-EET-induced pulmonary vasoconstriction is enabled by PASMC depolarization, which occurs in hypoxia. The discovery of the dual role of 5,6-EET in the regulation of pulmonary vascular tone may provide a basis for the development of novel therapeutic strategies for treatment of HPV-related diseases.

Insufficient Efficacy of Corpus Callosotomy for Epileptic Spasms With Biphasic Muscular Contractions.

Corpus callosotomy (CC) is the surgical strategy for drug-resistant epileptic seizures including epileptic spasms (ES). In this study we report a subtype of ES which is accompanied by two consecutive muscular contractions. This subtype has not been previously classified and may emerge via a complex epileptic network. We named these seizures “epileptic spasms with biphasic muscular contractions (ES-BMC)” and analyzed the association between them and CC outcomes. We enrolled 17 patients with ES who underwent CC before 20 years of age, and analyzed the records of long-term video-electroencephalogram (EEG) recordings. The outcomes of CC were ES-free (Engel's classification I) in 7 and residual ES (II to IV) in 10 patients. We statistically analyzed the associations between the presence of preoperative ES-BMC and the outcomes. Ages at CC ranged from 17 to 237 months. We analyzed 4–44 ictal EEGs for each patient. Five patients presented with ES-BMC with 6–40% of their whole ES on the presurgical video-EEG recordings, and all of them exhibited residual ES outcomes following CC. A Fisher's exact test revealed a significant positive correlation between the presence of preoperative ES-BMC and persistence of ES following CC (p = 0.044, odds ratio = 15.0, risk ratio = 2.0). The presence of ES-BMC may be useful in the presurgical prediction of CC outcomes in patients with ES.

A118 GUT MICROBIOTA TRANSPLANTATION FROM A PATIENT WITH SEVERE CONSTIPATION INDUCED CHANGES IN COLONIC FUNCTION AND STRUCTUR OF GNOTOBIOTIC MICE

Abstract Background Increasing evidence suggests that gut microbiota play a key role in gastrointestinal (GI) tract function. We have previously shown that fecal microbiota transplantation diarrhea predominant IBS patients into germ-free mice induces faster GI transit, increased permeability and innate immune activation. However, it is unknown whether gut dysfunction is induced by microbiota from patients with chronic constipation. Aims Here, we investigated the role of the intestinal microbiota in the expression of severe slow transit constipation in a patient with previous C difficile infection and extensive antibiotic exposure. Methods Germ-free (GF) mice (14 weeks old) were gavaged with diluted fecal content from the patient with constipation (PA) or a sex and age-matched healthy control (HC). 12 weeks later, we assessed gut motility and GI transit using videofluoroscopy and a bead expulsion test.. We then investigated intestinal and colonic smooth muscle isometric contraction in vitro using electric field stimulation (EFS), and acetylcholine (Ach) release was assessed by superfusion using [3H] choline. Histological changes were evaluated by H&E and immunohistochemistry. Results Mice with PA microbiota had faster whole GI transit (score 18.9 ± 0.9 (N=9) than mice with HC microbiota (15.4 ± 1.0, N=10, p=0.032), with markers located mainly in the distal small bowel and cecum. However, bead expulsion from the colon was significantly longer in PA mice (420.8 s ± 124.6 s, N=9) than in HC mice (82.6 s ± 20.0 s, N=10, p=0.026). This delayed colonic transit was likely due to colonic retroperistalsis visualized videofluoroscopically by retrograde flow of barium in the right colon of PA mice. There was no difference between the two groups in small intestinal or colonic tissues in Ach release or contractility induced by carbachol or KCl,. EFS caused transient biphasic relaxation and contraction in small intestine and colon, with the colonic contraction being stronger in the PA group. Microscopic tissue analysis showed disruption of the interstitial cells of Cajal (ICC) network and increased lymphocyte infiltration in colonic mucosa and submucosa in PA mice. Conclusions These results indicate that the microbiota is a driver of delayed colonic transit in a patient whose constipation started following extensive antibiotic exposure for C. difficile infection. The observed dysmotility pattern was not due to lower muscle contractility but likely caused by immune mediated changes in the ICC network. Funding Agencies CIHR

The effects of varying Mg2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

383: Human uterine contractility at term in relation to parity

It is well described that parous women perform better in labor than primigravid women, with less dystocic labour, shorter durations of labor and lower cesarean rates. There are no published data to our knowledge, comparing myometrial contractile parameters of nulliparous with parous women. Our study aim was to examine certain parameters in human myometrial tissue, and compare the results between the following 3 groups: Primigravid women (P0); Women with 1 previous delivery (P1); Women with more than 1 previous delivery (P>1); to determine if there was any difference between the contractile performance of each group. Myometrial biopsies were obtained at cesarean delivery (CD), at term, from n=74 women, with Institutional Ethical Review Board approval, Galway University Hospital. Each biopsy was dissected into 8 strips and suspended for in vitro tissue baths under physiological conditions, as previously described [1], giving a total of 592 samples. The following parameters of contractile performance were measured for spontaneous contractions: maximal amplitude (MAMP), mean contractile force (MCF), time to maximal amplitude, maximum rate of rise, frequency and occurrence of simple and complex (biphasic and multiphasic) contractions. Comparisons were made across the 3 groups: P0 n=14; P1 n=37; P>1 n=23. Statistical analysis was performed using T-Test Assuming Unequal Variance. The results obtained for the contractile parameters, for the 3 groups, are shown in Table 1. The MCF was greater for myometrial contractions in parous women (PO V P1 P=0.0061; P1 V P>1 P= 0.00089). Women P>1 displayed a shorter time to onset of first contraction (P=0.00046). For parous women there was a slower rate of rise and slower rate of relaxation. No difference was observed in any of the other parameters. (Table 2) These results suggest that there is an inherent biological difference between the contractile parameters of human myometrium in the third trimester, between primigravid and parous women. These findings correlate with clinical observations that parous women have more efficient and shorter labours. The increased force of contractions observed with parity, highlights the need for caution in these women undergoing augmentation of labor.

584: Human uterine contractility at term in relation to previous cesarean section

The efficiency of myometrial contractility in labor, for the woman who have had a previous cesarean delivery (CD), is an important factor for women undergoing trial of labor after a cesarean. There are no data, to our knowledge, on this topic. The aim of this study was to examine a range of contractile parameters in human myometrial tissue in vitro, and compare the results obtained between the following 3 groups: Women with no previous CD (CD=0); Women with 1 previous CD (CD=1); Women with greater than one CD (CD=>1). Myometrial biopsies were obtained at CD, performed at term, from n=74 women, with Institutional Ethical Review Board approval, Galway University Hospital. Myometrial strips were dissected into 8 uniform samples, and suspended for in vitro tissue bath analysis under physiological conditions, as previously described [1]. The following parameters of contractile performance for spontaneous contractions were measured: maximal amplitude (MAMP), mean contractile force (MCF), time to maximal amplitude, maximum rate of rise, frequency and occurrence of simple and complex (biphasic and multiphasic) contractions. Comparisons were made across the 3 groups as follows: CD=0 n=22; CD=1 n=37; CD=>1 n=15. Statistical analysis was performed using T-Test Assuming Unequal Variance. The results obtained for the different contractile parameters, for the 3 groups, are shown in Table 1. 592 tissue samples were analysed from 74 women. The only significant difference (Table 2) in contractile parameters found was in the frequency of contractions, with a lower frequency of contractions in the CD=1 group compared to CD=0 and CD=>1 groups (p=6.36E-06 and p=0.000135 respectively). These data indicate that there is no significant difference in the general biological characteristics of human uterine contractions in vitro, in women who have had one, or greater that one previous CD, in comparison to women who have had none. The only notable feature is that the frequency of contractions was greater in the sub-group of women who had one previous CD. These findings indicate that factors other than the functional contractility of uterine tissue, may be responsible for the increased CD rate observed among women undergoing VBAC.

Trypsin induces biphasic muscle contraction and relaxation via transient receptor potential vanilloid 1 and neurokinin receptors 1/2 in porcine esophageal body

Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1μM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK1) and NK2 antagonists, but not by an NK3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK1, NK2 and NK3) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK1 and NK2 antagonists, but not by the NK3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK1/2. Gap junctions were indispensable in this tachykinin-induced response.

Maternal Age and Contractility of Human Myometrium in Pregnancy.

There is controversy as to whether maternal age exerts an influence on the contractility of human myometrium in pregnancy. The aim of this study was to examine a series of functional contractile parameters of human myometrium in vitro, over a broad range of maternal ages. Myometrial tissue specimens were obtained at cesarean delivery from 32 women with maternal ages ranging from 28 to 52 years. Using in vitro recordings, a number of contractile parameters including maximal amplitude, mean contractile force, time to maximal amplitude, maximum rate of rise, and occurrence of simple and complex (biphasic and multiphasic) contractions were examined for spontaneous and induced contractile activity. The relationship between maternal age and individual parameters was evaluated using linear regression analysis. For all contractile parameters examined, for both spontaneous and induced contractions, no significant correlation was observed with maternal age between 28 and 52 years. The mean maximum amplitude values for spontaneous and oxytocin-induced contractions were 23 ± 3 and 43 ± 5 mN, respectively. The mean contractile forces for spontaneous and oxytocin-induced contractions were 1.5 ± 0.2 and 6.5 ± 0.9 mN, respectively. There was no variation in the proportion of biphasic or multiphasic contractions with maternal age. These results indicate there is no significant functional impairment of uterine contractility and no lack in responsiveness of myometrium in vitro, in the older mother. These findings do not support the concept that there may be a biological basis for dysfunctional labor or increased cesarean delivery rates in older parturients.

Regulation of nerve-evoked contractions of rabbit vas deferens by acetylcholine.

Stimulation of intramural nerves in the vas deferens of many species yields a classical biphasic contraction comprised of an initial fast component, mediated by P2X receptors and a second slower component, mediated by α1-adrenoceptors. It is also recognized that sympathetic nerve-mediated contractions of the vas deferens can be modulated by acetylcholine (Ach), however there is considerable disagreement in the literature regarding the precise contribution of cholinergic nerves to contraction of the vas deferens. In this study we examined the effect of cholinergic modulators on electric field stimulation (EFS)-evoked contractions of rabbit vas deferens and on cytosolic Ca2+ levels in isolated vas deferens smooth muscle cells (VDSMC). The sustained component of EFS-evoked contractions was inhibited by atropine and by the selective M3R antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP). EFS-evoked contractions were potentiated by Ach, carbachol (Cch), and neostigmine. The sustained phase of the EFS-evoked contraction was inhibited by prazosin, an α1-adrenoceptor antagonist and guanethidine, an inhibitor of noradrenaline release, even in the continued presence of Ach, Cch or neostigmine. The soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one enhanced the amplitude of EFS-evoked contractions and reduced the inhibitory effects of 4-DAMP. Isolated VDSMC displayed spontaneous Ca2+ oscillations, but did not respond to Cch. However, the α1-adrenoceptor agonist, phenylephrine, evoked a Ca2+ transient and contracted the cells. These data suggest that EFS-evoked contractions of the rabbit vas deferens are potentiated by activation of M3 receptors and reduced by activation of a sGC-dependent inhibitory pathway.

Ultrasonographic assessment of reticuloruminal motility in 45 cows.

The goal of this study was to investigate the feasibility of ultrasonographic assessment of reticuloruminal motility in 45 healthy cows. The transducers of five ultrasound machines were connected to a digital video recorder and placed simultaneously at five sites on the left side of the cows to scan the reticulorumen (reticulum; ruminal atrium; dorsal sac of the rumen; left longitudinal groove; ruminal recess, caudodorsal and caudoventral blind sacs). The video streams from all five ultrasound machines were recorded synchronously with the same time line and displayed on a single monitor. Time 0 was defined as the start of a biphasic reticular contraction. The reticulum was visualised in all cows and had 11.0 ± 2.12 biphasic contractions in 9 min. The ruminal atrium was visualised in 40 (89%) cows and had 10.7 ± 2.10 contractions in 9 min, which started at the time point 5.0 ± 0.83 sec and lasted 7.0 ± 2.14 sec. Contractions of the dorsal sac of the rumen, visible in all cows, were visualised in 29 (64%) cows. There were 9.5 ± 1.8 contractions in 9 min that started at the time point 4.0 ± 0.85 sec and lasted 8.2 ± 1.04 sec. The left longitudinal groove was seen contracting in 39 (87%) cows. There were 10.2 ± 1.98 contractions in 9 min that started at the time point 4.1 ± 1.81 sec and lasted 7.8 ± 1.19 sec. Contractions of the ventral sac of the rumen (ruminal recess) were seen in 31 (69%) cows. There were 7.5 ± 2.59 contractions in 9 min that started at the time point 14.3 ± 4.30 sec. Contractions of the caudodorsal and caudoventral blind sacs were seen in 34 (76%) cows. There were 9.0 ± 2.75 (1.0 ± 0.31) contractions/min and 9.4 ± 2.09 (1.0 ± 0.23) contractions/min of the dorsal and ventral blind sacs, and they started at the time points 6.2 ± 1.32 sec and 21.3 ± 6.20 sec, respectively. Primary contraction cycles were seen in all cows and secondary cycles in 22 (49%) cows. The former were complete in 37 (82%) cows and incomplete in 8 (18%). There were 11.0 ± 2.12 primary and 4.5 ± 2.15 secondary cycles in 9 min, and the ratio between primary and secondary cycles averaged 2.4:1. Ultrasonography is suitable for the assessment of reticuloruminal motility

Effects of Silodosin and Tamsulosin on the Seminal Vesicle Contractile Response.

To understand the mechanisms underlying ejaculation dysfunction caused by α1A-adrenocetor (AR) antagonists, the effects of α1A-AR antagonists on the contractile responses of the seminal vesicle were investigated. Isolated seminal vesicles from guinea pigs were cannulated and pressurized, and the changes in the intraluminal pressure were recorded. Periodic applications of electrical stimulation (ES) caused biphasic increase in the intraluminal pressure, that is, initial and subsequent contractions. The effects of silodosin and tamsulosin, α1A-AR antagonists, on the contractile responses were examined. The ES-induced biphasic contractions were blocked by tetrodotoxin (TTX). Silodosin and tamsulosin suppressed the initial contractions in a dose-dependent manner, while also exerting various inhibitory effects on the subsequent contractions. Increases in the intraluminal pressure facilitated spontaneous phasic contractions. The spontaneous contractions were not affected by TTX or α1A-AR antagonists, but were abolished by nifedipine. The initial contractions triggered by neuronal excitations were suppressed by silodosin and tamsulosin, suggesting that the ejaculation dysfunction may be attributed to the α1A-AR antagonist-mediated suppression of nerve-evoked contractions in the seminal vesicle. The subsequent contractions may be induced by mechanical stimulation associated with the initial, nerve-evoked contractions. Alternatively, other transmitters may be involved to various degrees in the neuromuscular transmission of the seminal vesicle.

L-type Ca2+ channel blockers inhibit the window contraction of mouse aorta segments with high affinity

L-type calcium channel blockers (LCCBs) reduce blood pressure more effectively in hypertensive than in normotensive subjects and are more effective in vascular smooth muscle (VSM) than in cardiac muscle. This has been explained by the depolarized resting potential of VSM in comparison with heart muscle cells and during hypertension, because both favor the “high affinity” inactivated state of the L-type calcium channel (LCC). Depolarized resting potentials, however, also increase Ca2+ influx via window, non-inactivating LCC. The present study investigated whether these channels can be effectively blocked by nifedipine, verapamil or diltiazem, as representatives of different LCCB classes. C57Bl6 mouse aortic segments were depolarized by 50mM K+ to attain similar degree of inactivation. The depolarization evoked biphasic contractions with the slow force component displaying higher sensitivity to LCCBs than the fast component. Removal of the fast force component increased, whereas stimulation of Ca2+ influx with the dihydropyridine BAY K8644, a structural analog of nifedipine, decreased the efficacy of the LCCBs. Addition of LCCBs during the contraction caused concentration-dependent relaxation, which was independent of the presence of a fast force component, but still showed lower sensitivity in the presence of BAY K8644. Our data suggest that steady-state contractions by depolarization with 50mM K+ are completely due to window Ca2+ influx, which is preferentially inhibited by LCCBs. Furthermore, results point to interactions between the LCCB receptors and Ca2+ ions or BAY K8644. The high affinity for open, non-inactivating LCC may play a dominant role in the anti-hypertensive effects of LCCBs.

In vivo kinematics of medial unicompartmental osteoarthritic knees during activities of daily living

Few studies exist describing unicompartmental osteoarthritic knee kinematics. Moreover, the role of the anterior cruciate ligament (ACL) in the determination of knee kinematics has not been fully described. The objective of the current study was to analyze the in vivo kinematics of knees with medial osteoarthritis (OA) and intact ACL during closed and open chained motion. Eight patients scheduled for UKA diagnosed with primary medial OA underwent knee CT-scans and video-fluoroscopy. Fluoroscopic analysis included stair climbing, chair rising and leg extension. Three-dimensional bone positions were obtained from each image by iterative procedures using a CAD-model-based shape-matching technique. Patterns of axial rotation and anterior-posterior (AP) motion of the medial and lateral femoral condyle were obtained with specific software. The femur reported an overall external rotation relative to the tibia from extension to flexion in all tasks. Average AP translation of the medial femoral condyle were smaller in open-chained tasks than in weight-bearing conditions. Average AP motion of the lateral femoral condyle reported an overall posterior translation with knee flexion. The absent natural "screw-home" mechanism and the lack of medial condyle posterior translation was explained by bone-cartilage defects and meniscal degeneration. Relevant findings were the kinematic pattern differences between weight-bearing and open chained activities, suggesting that in biphasic muscle contraction and unloaded conditions, the function of the cruciate ligaments was not physiological. The kinematics of knees with medial OA and intact ACL differed from healthy knees.

Ultrasonography of normal reticulum in 30 healthy buffalo (Bubalus bubalis)

Reticulum is incriminated in most cases of gastrointestinal disturbances in bovines and should be firstly examined. The aim of the present study was to assess ultrasonographic appearance and relation of reticulum to its surrounding in a normal buffalo (N = 30). Sonographic examination was performed in a standing nonsedated buffalo using 3.5-MHz convex transducer. B and M modes of display were used for examination. Reticulum appeared as a half-moon-shaped structure with a smooth contour; it could be easily accessed from the left and right ventral thoracic region than from midline. Biphasic contraction was recorded in all examined buffalo while only four animals had a triphasic contraction. Four to five contractions were recorded during a 4-minute examination period. The mean distance between reticulum and left abdominal wall was 1.603±0.566 cm. The duration of the first and second reticular contractions were 3.080±0.43 and 4.407±0.796 sec, respectively. However, the duration of relaxation interval was 42.63±3.187 sec. The amplitude of first contraction was 6.341±1.600 cm. In conclusion, reticular appearance and characters of reticular motility in buffalo is similar to that reported in cows, but reticulum was best accessed from left and right ventral thorax than from midline. M mode of display is an alternative tool for evaluation of reticular motility. The result of the present study could be used as a reference for reticular examination in a healthy buffalo.

P01 Mechanisms of hydrogen sulfide mediated contraction in rat small pulmonary arteries

The gas H2S (hereafter ‘sulfide’) typically acts as an vasodilator in the systemic circulation, but in pulmonary arteries elicits a biphasic contraction via an unknown mechanism. This is potentially a physiologically important effect, since it has also been proposed that the concentration of sulfide increases in pulmonary arteries under hypoxic conditions, thereby acting as an oxygen sensor which is important for initiating (and possibly mediating) hypoxic pulmonary vasoconstriction. We therefore assessed the mechanism of the sulfide-induced contraction in rat 2nd order pulmonary arteries (PA), using NaHS as a source of sulfide. Techniques used included recording of isometric force development and mitochondrial membrane potential in PA rings mounted in a small vessel myograph and measurement of reactive oxygen species (ROS) levels in segments of PA using the luminescent ROS indicator LO12 (10 μM). NaHS (500 μM) evoked a complex PA contraction comprising a small contraction followed by a second and larger contraction which gradually relaxed. The contraction was abolished by the mitochondrial complex three blocker antimycin (10 μg/ml) and was greatly reduced by the anti-oxidant TEMPOL (3 mM), the RyR blocker dantrolene (50 μM,) or the Rho kinase blocker Y27632 (1 μM). On the othe hand, the sulfide contraction was little affected by treatment of arteries with the mitochondrial complex 1 blocker rotenone (1 μM) or by their incubation in Ca2+-free PSS containing 0.2 mM EGTA. Incubation of pulmonary arteries in NaHS also caused activation of the monomeric G protein RhoA (measured using a G-lisa assay) and also of its downstream effector rho kinase (as shown by an increase in MYPT phosphorylation). A 15 min incubation of control cultured pulmonary artery smooth muscle cells (PASMC) in 1 mM NaHS (1 mM), followed by a 10 min wash to remove NaHS from the solution, led to a significant increase in cellular ROS production recorded using LO12. This increase in ROS was not present in PASMC in which expression of the the mitochondrial membrane flavoprotein sulfide-quinone oxoreductase (SQR) had been knocked down using siRNA. Application of a range of concentrations (10–1000 μM) of NaHS to pulmonary arteries led to a transient depolarisation of the mitochondrial membrane potential (recorded using TMRE) which was variably preceded and/or followed by hyperpolarisation. In the presence of rotenone, which caused depolarisation of the mitochondrial potential, low concentrations of NaHS (10–100 μM) caused a transient repolarisation, whereas high concentrations caused a complex response consisting of a transient repolarisation followed by a depolarisation and a second long-lasting repolarisation. Based on these results, we conclude that sulfide causes contraction by causing Ca2+ sensitisation and the release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors. Furthermore, we speculate that these responses are mediated by ROS produced by complex 3 owing to an increased flow of electrons into the mitochondrial electron transport chain consequent upon the metabolism of sulfide by SQR.

Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2) in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA

We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser188.

Tu1829 Trypsin Induced BiPhasic Contraction and Relaxation in the Porcine Lower Esophageal Sphincter

Tu1829 Trypsin Induced BiPhasic Contraction and Relaxation in the Porcine Lower Esophageal Sphincter

Ultrasonographic examination of the reticulum, rumen, omasum and abomasum during the first 100 days of life in calves

The goal of this study was to examine the development of the reticulum, rumen, omasum and abomasum in six calves from birth to 100days of age by means of six serial ultrasonographic examinations. The examinations were carried out in standing animals using a 5MHz-transducer as described previously. The calves were primarily fed milk until examination 4 and then they were weaned. The reticulum was assessed for its shape and contractility, the rumen for its size and content, the omasum for its size, content and motility and the abomasum for its size and content before and after the ingestion of milk. The reticulum was seen in all calves starting at examination 2 and had biphasic and triphasic contractions; the latter were associated with eructation. The rumen was always imaged in all calves as early as Day 1 and its visible size increased progressively in all intercostal spaces (ICSs) during the study period. The omasum was best imaged in the 8th or 9th ICS; it was seen medial to the liver dorsally and usually medial to small intestines ventrally. Its visible size in these two ICSs increased progressively but omasal motility was not apparent. In newborn calves the abomasum was the largest compartment and dominated the abdominal cavity. It was visible from the 5th ICS to the flank. Except for examination 2, the mean visible abomasal length was significantly larger after feeding than before. Lateral abomasal extension to the left was greater than to the right at examinations 1–4, but was much smaller than to the right at examinations 5 and 6 because of progressive expansion of the rumen. Abomasal extension into the right hemiabdomen changed little during the study period.