Transient Contraction Research Articles

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (≤ 3 μg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (≥ 10 μg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of α- and β-toxins with neuronal sodium channels.

Monitoring contractility in cardiac tissue with cellular resolution using biointegrated microlasers

The contractility of cardiac cells is a key parameter that describes the biomechanical characteristics of the beating heart, but functional monitoring of three-dimensional cardiac tissue with single-cell resolution remains a major challenge. Here, we introduce microscopic whispering-gallery-mode lasers into cardiac cells to realize all-optical recording of transient cardiac contraction profiles with cellular resolution. The brilliant emission and high spectral sensitivity of microlasers to local changes in refractive index enable long-term tracking of individual cardiac cells, monitoring of drug administration, accurate measurements of organ-scale contractility in live zebrafish, and robust contractility sensing through hundreds of micrometres of rat heart tissue. Our study reveals changes in sarcomeric protein density as an underlying factor to cardiac contraction. More broadly, the use of novel micro- and nanoscopic lasers as non-invasive, biointegrated optical sensors brings new opportunities to monitor a wide range of physiological parameters with cellular resolution. The incorporation of microsphere lasers into heart cells allows all-optical recording of cardiac contraction with cellular resolution. [This summary has been amended from ‘microdisk’ to ‘microsphere’ lasers.]

0027 Alternations in Contractility, Calcium Signaling and Nuclear Factor Kappa B Activation in Stem Cells Derived Cardiomyocytes Following Exposure to Obstructive Sleep Apnea Children’s Serum, and Intermittent Hypoxia

Abstract Introduction There are cardiovascular morbid effects of OSA in adults and children. We have previously reported over-expression of Nuclear-Factor-κappa B in tonsils of children with OSA, and NF-κB activation after exposure to sera of children with OSA. In order to investigate NF-kB activation and the physiology of cardiomyocytes of human origin (CM’s), we have established an ex-vivo model where CM’s derived from human embryonic stem cells H-9.1 clone (hES) and from Induced pluripotent stem cells (iPSC) are either incubated with human sera, or exposed to intermittent hypoxia(IH) or room air (RA). Methods Serum samples were drawn following overnight polysomnography (PSG) from children with OSA (AHI>5) or control (AHI<1). Differentiated human cardiomyocytes were incubated with 5% OSA sera or control. Average cell beating/min was determined and NF-kB p50 and p65 cytoplasmic or nuclear localization were detected by immunofluorescence (operetta). CM’s were exposed to either RA or to IH (21% alternating with 1% O2, 6 cycles/h, 24 h) in a chamber (BioSpherix Instruments, Redfield, NY)Intracellular calcium transient signal (ionoptix) and mechanical contraction of CM (Musclemotion) were assessed. Results 1. Cell beating/min was reduced in cells incubated with OSA (n=10) as compared to control (n=10);(p<0.01), and following IH exposure vs RA (p<0.01),). 2. There is an over-expression of NF-κB p50, p65 subunits as a result of exposing CMs to OSA sera, and following IH exposure 3. a rapid decrease (up to 100%) was measured in calcium transient amplitude as well as contraction amplitude (p<0.05) after adding OSA sera, that was completely restored after washing. Conclusion We revealed human cardiomyocytes NF-kB activation and decreased contractility following exposure to OSA sera and to IH. The rapid decrease in calcium signaling and contractility infer the presence of factors that reversibly affects CMs contraction. This ex-vivo model enables the study of functional and molecular alterations in human CM Support Israel science Foundation (ISF) 1344/15

Lower Ca2+ enhances the K+-induced force depression in normal and HyperKPP mouse muscles.

Hyperkalemic periodic paralysis (HyperKPP) manifests as stiffness or subclinical myotonic discharges before or during periods of episodic muscle weakness or paralysis. Ingestion of Ca2+ alleviates HyperKPP symptoms, but the mechanism is unknown because lowering extracellular [Ca2+] ([Ca2+]e) has no effect on force development in normal muscles under normal conditions. Lowering [Ca2+]e, however, is known to increase the inactivation of voltage-gated cation channels, especially when the membrane is depolarized. Two hypotheses were tested: (1) lowering [Ca2+]e depresses force in normal muscles under conditions that depolarize the cell membrane; and (2) HyperKPP muscles have a greater sensitivity to low Ca2+-induced force depression because many fibers are depolarized, even at a normal [K+]e. In wild type muscles, lowering [Ca2+]e from 2.4 to 0.3 mM had little effect on tetanic force and membrane excitability at a normal K+ concentration of 4.7 mM, whereas it significantly enhanced K+-induced depression of force and membrane excitability. In HyperKPP muscles, lowering [Ca2+]e enhanced the K+-induced loss of force and membrane excitability not only at elevated [K+]e but also at 4.7 mM K+. Lowering [Ca2+]e increased the incidence of generating fast and transient contractures and gave rise to a slower increase in unstimulated force, especially in HyperKPP muscles. Lowering [Ca2+]e reduced the efficacy of salbutamol, a β2 adrenergic receptor agonist and a treatment for HyperKPP, to increase force at elevated [K+]e. Replacing Ca2+ by an equivalent concentration of Mg2+ neither fully nor consistently reverses the effects of lowering [Ca2+]e. These results suggest that the greater Ca2+ sensitivity of HyperKPP muscles primarily relates to (1) a greater effect of Ca2+ in depolarized fibers and (2) an increased proportion of depolarized HyperKPP muscle fibers compared with control muscle fibers, even at normal [K+]e.

Histamine may directly contract urinary bladder smooth muscle

Mast cell degranulation and histamine release contribute to painful bladder syndrome, urinary tract infections, interstitial cystitis and other bladder disorders. The effects of histamine are well‐studied in other tissues, but little is known about the role of histamine and histamine receptors within the bladder. Since other smooth muscle cells express H1 (contractile) and H2 (relaxant) histamine receptors, we tested the hypothesis that histamine causes contraction of the urinary bladder smooth muscle in mice. All procedures followed institutional guidelines and were approved by the Institutional Animal Care and Use Committees of Michigan State University. Six to 12‐week‐old C57Bl/6 mice were euthanized and dissected bladders were cut into approximately 2 mm wide bladder strips, with or without the urothelium attached, for isometric contractility experiments. Both histamine H1 and H2 receptors were expressed in bladder smooth muscle cells. Increasing concentrations of histamine (100 nM – 300 μM) did not contract strips without urothelium and minimally contracted bladder strips with urothelium. However, bolus administration of 200 μM histamine caused a rapid but transient contraction in strips with and without urothelium. The H1 receptor blocker fexofenadine (5 – 10 μM) blocked these contractions (P<0.05; N=5), whereas the H2 receptor blocker cimetidine (5 – 10 μM) had no effect. In addition, transient receptor potential vanilloid 1 (TRPV1) channel blocker capsazepine significantly reduced histamine‐induced contractions only in bladder strips without urothelium, whereas the Na+ channel blocker tetrodotoxin (TTX) had no effect in any tissue (P < 0.05; N= 3–5). The mast cell activator compound 48/80 (10 μg/ml) contracted urinary bladder smooth muscle, and this contraction was also unaffected by TTX. These results indicate that H1 receptors on urinary bladder smooth cells likely modulate histamine‐induced bladder contraction, and sufficient amounts of histamine can be released from mast cells to cause a contraction.Support or Funding InformationThis research is supported by NIH K01‐DK103840 (NRT) and NIH R01‐DK119615 (NRT & GCM).

Smooth Muscle Tone Contributes to Bladder Wall Stiffness during Filling

Sensations of bladder fullness are conveyed to the central nervous system by sensory nerves as a response to distention of the urinary bladder. This distention is accompanied by non‐voiding transient pressure events and contractions called “micromotions” that are responsible for the majority of sensory outflow during bladder filling. Changes in smooth muscle contractility along with alterations in wall stiffness and sensory feedback are observed in most UB pathologies. Thus, the mechanical properties of bladder wall may play a crucial role in communicating a sense of bladder fullness. We hypothesize that the contractile apparatus of the urinary bladder is actively involved in maintaining bladder wall stiffness during filling.Whole mouse bladders were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system, for simultaneous measurement of bladder wall micromotions, bladder volume, intravesical pressure, bladder wall stress, and bladder wall strain during ex vivo bladder filling. Bladders were filled at a constant rate (30 μl/min) until intravesical pressure reached 25 mmHg. During filling, bladder wall micromotions led to transient pressure events (0–2 mmHg in amplitude). These micromotions began on the lateral sides of the bladder wall near the trigone and expanded upwards as the bladder filled. Baseline bladder pressure, however, remained relatively low over a large infused volume (~350 μL). Next, we derived the stress versus strain relationship of the bladder wall from the pressure‐volume recordings during filling. At baseline pressures >15 mmHg, intravesical pressure rapidly increased and transient pressure events could no longer be distinguished implying that wall compliance is maintained until larger infused volumes, after which bladder wall stress with respect to strain increases rapidly. To determine if smooth muscle tone altered bladder wall stress during filling, bladder was exposed to myosin inhibitor blebbistatin (10 μM) or PI3 kinase inhibitor wortmannin (10 μM). Both blebbistatin and wortmannin effectively diminished the micromotions and shifted the stress vs strain curve to the right, indicating that the smooth muscle contractile apparatus and smooth muscle tone contribute to bladder wall stiffness, but predominantly only at higher filling volumes. These findings suggest that smooth muscle tone directly increases bladder wall stiffness and that changes in this tone could alter sensory feedback during bladder filling.Support or Funding InformationSupported by NIH K01DK103840 (NRT) and R01DK119615 (NRT).

Pimpinella anisum extract attenuates spontaneous and agonist-induced uterine contraction in term-pregnant rats

Ethnopharmacology relevancePimpinella anisum is a well-known traditional medicinal herb which has been used in folk medicine as an antiulcer, anticancer, antibacterial and as a muscle relaxant. Aim of the studyThis study was performed to explore the modulatory effects of Pimpinella anisum on term-pregnant rat uterine contractility and to investigate its possible underlying mechanisms. Material and methodsIntact uterine strips without endometrial layer were isolated from female term-pregnant Wistar rats (22 days of gestation) and mounted in a tissue bath apparatus for in vitro isometric force recording. The effects of different concentrations of Pimpinella anisum extract (PAE) (1, 3, 5, and 7 mg/mL) were examined on uterine contractions generated spontaneously or induced with oxytocin (5 nmol/L), Bay K8644 (1 μmol/L), and carbachol (10 μmol/L). In some experiments, PAE was applied on depolarized myometrium in the presence of high-KCl solution (60 mmol/L). The effect on Ca2+ release was also examined. ResultsApplication of PAE significantly reduced uterine contractions generated spontaneously or induced with oxytocin, Bay K8644, and carbachol in a concentration-dependent manner (n = 7; P < 0.01). In depolarized myometrium, PAE significantly reduced the tonic force induced by high-KCl solution (n = 7; P < 0.01). PAE prevented oxytocin-induced transient contraction in the entire absence of external calcium (n = 7; P < 0.01). ConclusionThe present findings demonstrate the potentials of PAE to relax pregnant uterine contractions possibly by blocking Ca2+ entry via L-type calcium channels and inhibiting Ca2+ release from the internal store. The tocolytic effects of PAE may be a potential adjuvant against strong premature uterine contractions which threaten early pregnancy although clinical studies are required.

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&amp;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 the Quantum and Finesse Bridles on Equine M. Brachiocephalicus and M. Splenius Function at Three Different Speeds

Badly designed and fitted tack induces physiological stress responses in horses, and may compromise animal welfare. Moreover, horses, just like humans, comprise a series of interconnected myofascial lines. However, to date there are no measurements of the effects of horse tack, such as bridles on muscle parameters. This study used acoustic myography to test whether two commercial anatomically designed and fitted bridles, have a measurable and positive effect on both equine muscle-function and performance. A Quantum bridle was tested on 12 Icelandic horses, whilst a Finesse bridle was tested on 8 Icelandic horses, and results compared with data from a standard bridle (n = 12) tested at the same time and under identical conditions. Sensors were placed on M. Brachiocephalicus and M. Splenius, and the horses exercised following a set protocol at three speeds. The results revealed statistically significant improvements in muscle performance as assessed by both the regularity of the recording at each speed, and the number of transient powerful contractions (spikes) for the anatomically designed and fitted bridles, compared with the standard bridle. It is concluded that the effect of anatomically designed bridles can not only be measured in the neck muscles of exercising horses, but that they appear to have a positive effect on muscle performance.

Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels

BackgroundLocal injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment.MethodsThe intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3–4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50–75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant.ResultsAddition of 1.2% BaCl2 to PSS depolarized myofibers from − 79 ± 3 mV to − 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h.ConclusionsBaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.

Fast relaxation and desensitization of angiotensin II contraction in the pulmonary artery via AT1R and Akt-mediated phosphorylation of muscular eNOS.

Angiotensin II (AngII) triggers a transient contraction of pulmonary arteries (PAs) followed by protracted desensitization. Based on the unconventional eNOS expression in PA smooth muscle cells (PASMCs), we hypothesized that activation of smooth muscle eNOS by AngII might be responsible for fast relaxation and tachyphylaxis. Using dual-wire myograph, mechanically endothelium-denuded rat PA [E(-)PA] showed AngII concentration-dependent transient contractions (ΔTAngII, 95% decay within 1min), which were abolished by losartan (AT1R antagonist). Neither PD123319 (AT2R antagonist) nor A779 (MasR antagonist) affected ΔTAngII. When the vessels were pretreated with L-NAME (NOS inhibitor), ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), ΔTAngII of E(-)PA became larger and sustained, whereas nNOS or iNOS inhibitors had no such effect. Immunoblotting of human PASMCs (hPASMCs) also showed eNOS expression, and AngII treatment induced activating phosphorylations of Ser1177 in eNOS and of Ser473 in Akt (Ser/Thr protein kinase B), an upstream signal of eNOS phosphorylation. In addition, L-NAME co-treatment promoted AngII-induced Ser19 phosphorylation of myosin light chain. In hPASMCs, AngII abolished plasma membrane expression of AT1R, and recovery by washout took more than 1h. Consistent with the data from hPASMCs, the second application of AngII to E(-)PA did not induce contraction, and significant recovery of ΔTAngII required prolonged washout (> 2h) in the myography study. L-NAME treatment before the second application facilitated recovery of ΔTAngII. Muscular eNOS plays an auto-inhibitory role in ΔTAngII of PAs. The molecular changes investigated in hPASMCs revealed eNOS phosphorylation and internalization of AT1R by AngII. We propose that the rat PA smooth muscle eNOS-induced lusitropy and slow recovery of AT1R from tachyphylaxis might counterbalance the excessive contractile response to AngII, contributing to the distinctive low-pressure pulmonary circulation.

Bone Marrow T-Cell Sequestration and Lymphopenia Accompanying Ischemic Stroke are Mediated by T-Cell S1P1 Loss

Abstract INTRODUCTION Sequestration of T-cells in bone marrow is a phenomenon recently characterized by our group in the setting of intracranial tumors. Our findings suggest that it is the intracranial location rather than tumor histology that elicits this phenotype. Sequestration is accompanied by lymphopenia and lymphoid organ contraction and is mediated by loss of the S1P1 receptor from the T-cell surface. We now reveal that this phenomenon is not only unique to brain tumors, but accompanies additional intracranial pathologies, most notably ischemic stroke. METHODS Blood, bone marrow, and spleens were collected from mice at day 2, 5, 7, or 14 following stroke via middle cerebral artery occlusion or sham surgery and analyzed by flow cytometry. T-cell S1P1 levels were assessed, along with T-cell counts in each compartment. S1P1 receptor stabilization was achieved with a knock-in model that inhibited receptor internalization. RESULTS Following stroke induction, T-cells accumulated in the bone marrow of injured mice. T-cell numbers peaked at day 7 poststroke before returning to normal levels by day 14. Bone marrow accumulation was accompanied by transient T-cell lymphopenia and splenic contraction following stroke. T-cells in the bone marrow yielded decreased levels of S1P1 on their surface. Conversely, mice with genetically stabilized T-cell S1P1 were protected against sequestration, lymphopenia, and splenic contraction following stroke. CONCLUSION Bone marrow T-cell sequestration occurs transiently following stroke and is mediated by the S1P-S1P1 axis. This may prove to be an adaptive mechanism to limit intracranial inflammation following an initial insult. Better understanding of this phenomenon may uncover a novel mechanism of immune privilege and allow for therapeutic modulation in the setting of stroke, brain tumor, and other types of intracranial injury.

Properties of SK3 channel-expressing PDGFRα (+) cells in the rodent urinary bladder

Localisation of platelet-derived growth factor receptor-α (PDGFRα) (+) cells expressing small-conductance Ca2+-activated K+ (SK3) channels in the urinary bladder was investigated, while putative roles of SK3 (+) PDGFRα (+) cells in suppressing detrusor smooth muscle (DSM) spontaneous activity were explored. In guinea-pig bladder, immunohistochemistry for SK3 channels, PDGFRα or vimentin was examined, as were the effects of purinergic agonists on spontaneous phasic contractions (SPCs). In bladder of PDGFRα-GFP mice, the effects of purinergic agonists on intracellular Ca2+ signaling in PDGFRα (+) cells or DSM cells in situ and SPCs were investigated. SK3 (+) cells co-expressing PDGFRα or vimentin were distributed in DSM bundles but not inter-bundle spaces or lamina propria. SK3 (+) cells had a stellate- or spindle-shape cell body extending processes. MRS2365 (100 nM or 1 μM), a P2Y1 agonist, caused a transient contraction without inhibiting SPCs in both DSM and lamina propria. In PDGFRα-GFP mice bladder, MRS2365, (100 nM), ADP (100 μM) or ATP (100 μM) increased the Ca2+ level of PDGFRα (+) cells without suppressing spontaneous Ca2+ transients in neighboring DSM cells, and also failed to suppress SPCs. Preferential localisation of SK3 positive PDGFRα (+) cells in DSM bundles appears to indicate their functional interaction with DSM cells. However, increases in Ca2+ level of PDGFRα (+) cells upon purinergic stimulation are not associated with the inhibition of Ca2+ or contractile activity in DSM cells. Thus, it is unlikely that the SK3-dependent hyperpolarisation generated in SK3 expressing PDGFRα (+) cells is transmitted to DSMs to suppress their excitability.

Vasodilatory Effects and Mechanisms of Action of Bacopa monnieri Active Compounds on Rat Mesenteric Arteries.

B. monnieri extract (BME) is an abundant source of bioactive compounds, including saponins and flavonoids known to produce vasodilation. However, it is unclear which components are the more effective vasodilators. The aim of this research was to investigate the vasorelaxant effects and mechanisms of action of saponins and flavonoids on rat isolated mesenteric arteries using the organ bath technique. The vasorelaxant mechanisms, including endothelial nitric oxide synthase (eNOS) pathway and calcium flux were examined. Saponins (bacoside A and bacopaside I), and flavonoids (luteolin and apigenin) at 0.1–100 µM caused vasorelaxation in a concentration-dependent manner. Luteolin and apigenin produced vasorelaxation in endothelial intact vessels with more efficacy (Emax 99.4 ± 0.7 and 95.3 ± 2.6%) and potency (EC50 4.35 ± 1.31 and 8.93 ± 3.33 µM) than bacoside A and bacopaside I (Emax 83.6 ± 2.9 and 79.9 ± 8.2%; EC50 10.8 ± 5.9 and 14.6 ± 5.4 µM). Pretreatment of endothelial intact rings, with L-NAME (100 µM); an eNOS inhibitor, or removal of the endothelium reduced the relaxant effects of all compounds. In K+-depolarised vessels suspended in Ca2+-free solution, these active compounds inhibited CaCl2-induced contraction in endothelial denuded arterial rings. Moreover, the active compounds attenuated transient contractions induced by 10 µM phenylephrine in Ca2+-free medium containing EGTA (1 mM). Thus, relaxant effects occurred in both endothelial intact and denuded vessels which signify actions through both endothelium and vascular smooth muscle cells. In conclusion, the flavonoids have about twice the potency of saponins as vasodilators. However, in the BME, there is ~20 × the amount of vaso-reactive saponins and thus are more effective.

Activation of Cav1.2 and BKCa is involved in the downregulation of caffeine-induced contraction in mice mesenteric arteries

AimsCaffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. Main methodsSecond-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 μM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 μM) and ruthenium red (RuR; 10 μM), transient receptor potential (TRPs) channels blockers; capsazepine (10 μM) and HC067047 (10 μM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 μM), a selective BKCa blocker; and SKF-96365 (30 μM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. Key findingsThe TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SignificanceOur results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.

Involvement of different receptor subtypes in prostaglandin E2-induced contraction and relaxation in the lower esophageal sphincter and esophageal body

Prostaglandin E2 (PGE2) plays a role in the pathogenesis of gastro-esophageal reflux disease (GERD). There are 4 subtypes of PGE2, PGE2 receptor 1, 2, 3 and 4 (EP 1–4). In GERD patents, PGE2, EP2 and EP4 are upregulated. However, the effects of PGE2 on esophageal motility remain elusive.We examined how PGE2 regulates motility in the porcine circular smooth muscle of the lower esophageal sphincter (LES), and the circular and longitudinal smooth muscle of the esophagus body in organ bath.PGE2 induced tonic relaxation in the LES and circular smooth muscle, but transient contraction in longitudinal smooth muscle. The relaxation of the LES and circular smooth muscle was similar in pattern and mechanism, but was much larger in the LES. The relaxation was completely blocked by a voltage-gated K+ channel blocker or 40 mM K+ depolarization, indicating the involvement of K+ channel. Longitudinal smooth muscle contraction was completely blocked by an L-type Ca2+ channel blocker, showing the contribution of Ca2+ movement. The involvement of the EP receptor in motility was examined with selective receptor agonists and antagonists. Activation of EP2 and EP4 caused relaxation in the LES and circular smooth muscle. Compatible with PGE2, EP2 and EP4 agonists caused more significant relaxation in the LES than in circular smooth muscle. EP1 contributed to the longitudinal smooth muscle contraction.The different effects of PGE2 in the LES, circular and longitudinal smooth muscle contributes to esophageal motility, their impairment might increase the amount and frequency of esophageal reflux.

Targeting HIF-1α in combination with PPARα activation and postnatal factors promotes the metabolic maturation of human induced pluripotent stem cell-derived cardiomyocytes

Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca2+ transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.

Phasic, Rapidly‐Propagating Calcium Transients Occur in a Non‐Muscle Cell Network within the Mouse Urinary Bladder Wall

Multiple species, including humans, exhibit “non‐voiding” or “transient” contractions of the bladder wall during filling that are accompanied by large bursts of activity from bladder sensory nerves. This suggests that transient contractions play an important role as sensors of bladder fullness, but how transient contractions are generated remains unclear. Using genetically‐encoded Ca2+ indicator mice and novel macroscopic imaging techniques, we sought to determine what Ca2+ signals drive TC generation and coordination across the bladder wall. We utilized mice that constitutively express the Ca2+ indicators GCaMP5 or GCaMP6f as an expression‐dependent fate map of smooth muscle cells (Acta2 promoter) or non‐muscle cells (Trpv1 promoter) within the urinary bladder wall. We discovered that transient pressure events (TPEs) are actually the integration of numerous micro‐contractions of the bladder wall. These micromotions vary in position, frequency and duration, but result in regular, phasic increases in intravesical pressure. Ca2+ events in urinary bladder smooth muscle (UBSM) were restricted to small patches muscle cells and seemed to occur stochastically. Interestingly, we discovered a network of cells within the bladder wall of Trpv1‐GCaMP6f mice that were neither smooth muscle cells nor nerve fibers. This network exhibited extremely rapid and phasic Ca2+ transients that propagated quickly across the entire bladder wall, suggesting that the cells were electrically coupled. The slower waxing and waning of the high‐frequency Ca2+ transient intensity closely aligned with the frequency of TPEs. Surprisingly, this network of cells was unresponsive to the TRPV1 agonist capsaicin, suggesting that TRPV1 channels were no longer functional in these cells. These data suggest that the bladder wall micro‐motions underlying TPEs may be driven by phasic Ca2+ transients that occur in a non‐muscle cell network within the bladder wall.Support or Funding InformationSupported by NIH K01DK103840 (NRT) and R37DK053832 (MTN).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms underlying the vasorelaxant effect of trans-4-methoxy-β-nitrostyrene in the rat mesenteric resistance arteries

Mechanisms underlying the vasorelaxant effects of the synthetic nitro compound, trans-4-methoxy-β-nitrostyrene (T4MN) were studied in isolated small resistance arteries from spontaneously hypertensive rats (SHRs). T4MN caused vasorelaxation in endothelium-intact third-order branches of the mesenteric artery pre-contracted with noradrenaline (NA). This effect was unchanged by indomethacin and atropine but was significantly reduced by endothelium removal, L-NAME, LY294002, glybenclamide, TEA, apamin, TRAM 34, or by the association of apamin and TRAM 34. Pretreatment with the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the T4MN-induced relaxation in endothelium-intact, but not in denuded preparations. Incubation of small resistance arteries with T4MN increased nitric oxide (NO) production, an effect that was blocked by L-NAME. In Ca2+-free medium, T4MN inhibits the contractions induced by (i) NA, (ii) exogenous calcium through receptor- or voltage-operated Ca2+ channels and (iii) those evoked by Ca2+ influx through stores-operated Ca2+ channels activated by thapsigargin-induced Ca2+ store depletion. In contrast, T4MN was inert against the transient contraction induced by caffeine in Ca2+-free medium. In conclusion, T4MN induced effective vasorelaxant effects in isolated small resistance arteries from SHRs. This vasorelaxation seems to be mediated partly by an endothelium-dependent mechanism involving activation of Akt/eNOS/NO pathway and partly by an endothelium-independent mechanism through activation of sGC/cGMP/PKG pathway in vascular smooth muscle, leading to inhibition of Ca2+ influx from the extracellular milieu and IP3-sensitive intracellular Ca2+ release as well as activation of potassium channels.

Excitation-Contraction Coupling in Ureteric Smooth Muscle: Mechanisms Driving Ureteric Peristalsis.

The ureter acts as a functional syncytium and is controlled by a propagating plateau-type action potential (AP) which gives rise to a wave of contraction (ureteral peristalsis) via a process called excitation-contraction (E-C)coupling. The second messenger Ca2+ activates Ca2+/calmodulin-dependent myosin light chain kinase-dependent phosphorylation of 20-kDa regulatory light chains of myosin which leads to ureteric contraction. Ca2+ entry from the extracellular space via voltage-gated L-type Ca2+ channels (VGCCs) provides the major source of activator Ca2+, responsible for generation of both the AP and a Ca2+ transient that appears as an intercellular Ca2+ wave. The AP, inward Ca2+ current, Ca2+ transient and twitch contraction are all fully blocked by the selective L-type Ca2+ channel blocker nifedipine. Ca2+ entry via VGCCs, coupled to activation of Ca2+-sensitive K+ (KCa) or Cl- (ClCa) channels, acts as a negative or positive feedback mechanism, respectively, to control excitability and the amplitude and duration of the plateau component of the AP, Ca2+ transient and twitch contraction. The ureter, isolated from the pelvis, is not spontaneously active. However, spontaneous activity can be initiated in the proximal and distal ureter by a variety of biological effectors such as neurotransmitters, paracrine, endocrine and inflammatory factors. Applied agonists depolarise ureteric smooth muscles cells to threshold of AP activation, initiating propagating intercellular AP-mediated Ca2+ waves to produce antegrade and/or retrograde ureteric peristalsis. Several mechanisms have been proposed to describe agonist-induced depolarization of ureteric smooth muscle, which include suppression of K+ channels, stimulation of ClCa current and activation of non-selective cation receptor/store operated channels.