A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

Physiological Roles of Lymph Flow-Mediated Nitric Oxide in Lymphatic System.

It is known that nitric oxide (NO) is a gas and synthesized from l-arginine by the NO synthase (NOS) in vascular endothelial cells. The diffused NO activates the guanosine monophosphate, which initiates a series of intracellular events, leading to physiological response such as vasodilation. There are three different types of NOS, namely endothelial constitutive NOS (ecNOS), neuronal NOS (nNOS), and cytokine-inducible NOS (iNOS). The ecNOS and nNOS are expressed constitutively at low levels and can be activated rapidly by an increase in cytoplasmic calcium ions. In contrast, the iNOS is induced when macrophages are activated by cytokine, resulting in the induction of pathophysiological effects. Lymph flow is known to stimulate the release of NO from lymphatic endothelial cells (LEC) and then produce the relaxation of lymphatic smooth muscle cells. The NO also plays a key role in the control of lymphatic pump activity in vivo. Many studies have shown the NO-mediated findings in various kinds of lymph vessels. However, there is no or little study to demonstrate the effects of lymph flow on the molecular expression of ecNOS mRNA and the protein. In addition, little study is available for clarifying the relationship between NO and sympathetic nerve fibers in the regulation of lymph transport and production. Therefore, in this review, the experimental findings of lymph flow-mediated increases in the ecNOS mRNA and the protein in LEC are demonstrated in detail. In addition, the roles of NO and aminergic nerve fibers in the physiological control system of lymph transport and production are discussed.

Anoxia/reoxygenation enhances spontaneous contractile activity via TRPA1 channel and COX2 activation in isolated rat whole bladder.

PURPOSE: Bladder ischemia/reperfusion is an important etiologic factor for overactive bladder disease. The occurrence of this disease is closely associated with enhanced spontaneous contractile activity of the bladder. However, the relationship between bladder ischemia/reperfusion and altered spontaneous bladder contractions (SBC) remains poorly studied. Therefore, the present study investigated whether ischemia/reperfusion affects SBC ex vivo. METHODS: SBC was measured using isolated whole bladder preparations from rats. The preparations were exposed to anoxia (95% N2 ) for 0.5-6 h, followed by reoxygenation (95% O2 ) in Krebs medium. RESULTS: Anoxia followed by reoxygenation significantly enhanced the amplitude of SBC without affecting its frequency in an anoxic duration-dependent manner. The 5 h anoxia/reoxygenation-induced enhancement of SBC amplitude was completely suppressed by an antioxidant combination of L(+)-ascorbate/D, L-α-tocopherol, or N-acetyl cysteine. Additionally, the enhanced SBC amplitude was inhibited in a concentration-dependent manner by the nonselective TRP antagonist ruthenium red, or selective TRPA1 antagonists HC-030031 or AP-18. A similar inhibitory effect was obtained after repeated treatment with the TRPA1 agonist allyl isothiocyanate, as it induced acute desensitization of TRPA1 channels. Further, the enhanced SBC amplitude was significantly diminished by the nonselective cyclooxygenase (COX) inhibitor indomethacin or selective COX2 inhibitor NS-398, but not by the selective COX1 inhibitor SC-560 and 5-lipoxygenase inhibitor MK-886. CONCLUSIONS: The study findings reveal that the spontaneous contractile activity of the bladder is significantly enhanced in response to anoxia/reoxygenation, and that oxidative stress and activation of TRPA1 and COX2 (the resulting production of prostaglandins) are involved in the enhanced SBC activity.

Physical optimization of cell proliferation and differentiation using spinner flask and microcarriers.

The traditional breeding industry has been increasingly saturated and caused environmental pollution, disease transmission, excessive resource use, and methane emission; however, it still cannot meet the needs of the growing population. To explore other alternatives, researchers focused on cell agriculture and cell-based meat, especially large-scale cell culture. As a prerequisite for production, large-scale culture technology has become an important bottleneck restricting cell-based meat industrialization. In this study, the single-factor variable method was adopted to examine the influence of Cytodex1 microcarrier pretreatment, spinner flask reaction vessel, cell culture medium, serum and cell incubation, and other influencing factors on large-scale cell cultures to identify the optimization parameters suitable for 3D culture environment. Collagen and 3D culture were also prospectively explored to promote myogenesis and cultivate tissue-like muscle fibers that contract spontaneously. This research lays a theoretical foundation and an exploratory practice for large-scale cell cultures and provides a study reference for the microenvironment of myoblast culture in vitro, a feasible direction for the cell therapy of muscular dystrophy, and prerequisites for the industrialized manufacturing of cell-based meat. Graphical summary: Research on large-scale myoblast culture using spinner flasks and microcarriers. For cell culture, the microcarriers were pretreated with UV and collagen. Cell seeding condition, spinner flask speed, resting time, and spinner flask culture microenvironment were then optimized. Finally, two culture systems were prepared: a culture system based on large-scale cell expansion and a culture system for myogenesis promotion and differentiation.

Constitutively active HCN channels constrain detrusor excitability and modulate evoked contractions of human bladder.

OBJECTIVE: Expression of Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is reported in bladder, but the functional role remains unsettled. Here, we immunolocalized the HCN1 and HCN4 subtype in human bladder and investigated their functional significance. METHODS: Bladder procured from ten organ donors was dissected into mucosa (containing urothelium and submucosa) and detrusor for double immunofluorescence of HCN1 and 4 subtypes with gap junction and neural proteins together with isometric tension recordings. Mucosa intact and denuded detrusor strips were stretched to a basal tension of 10 mN for eliciting either tetrodotoxin (TTX) resistant spontaneous, carbachol evoked contractions and TTX sensitive electrical field stimulated (EFS), pre and post-addition of HCN blocker, ZD7288 or the activator, Lamotrigine or the cholinesterase inhibitor, Neostigmine. RESULTS: Double immunofluorescence revealed immunolocalization of HCN1 and HCN4 subtype with calcitonin gene related peptide (CGRP), choline acetyl transferase and gap junction proteins in mucosa and detrusor. Removal of mucosa significantly raised the resting tension and the force of spontaneous contractions upon cumulative addition of ZD7288 in micromolar range relative to Lamotrigine treated strips (P<0.05). ZD7288 [10 nM] did not affect the contractile response evoked by EFS or carbachol, but the addition of ZD7288 [10 nM] in presence of Neostigmine [1 µM] significantly enhanced the atropine and TTX sensitive EFS evoked contractions of mucosa denuded strips. CONCLUSIONS: Overall, HCN channels immunolocalized in mucosa, smooth muscle, gap junctions and nerve fibers exert a tonic constraint on detrusor excitability, enable spatio-temporal integration of evoked contractions and constrain the release of neurotransmitters, respectively. In contrast to the pacemaker role in other organs, findings argue for a non-pacemaking role of HCN channels in human bladder.

Justicia flava leaf extract potently relaxes pregnant human myometrial contractility: a lead plant for drug discovery of new tocolytic drugs.

NEW FINDINGS: What is the central question of this study? Can Justicia flava leaf extract (JF) inhibit human myometrial contractility as was previously shown in mouse myometrium? What is the main finding and its importance? JF abolished human myometrial contractions and therefore presents as a lead plant in drug discovery studies involving drugs for preterm birth. ABSTRACT: In the search for new potent therapies for preterm labour, Justicia flava leaf extract (JF) was previously shown to potently inhibit uterine contractility in both pregnant and non-pregnant mouse uterus. This study took the investigation a step further and investigated the activity of JF on pregnant human myometrial contractility. JF potently inhibited human myometrial contractility in a concentration-dependent manner. This pilot study provides evidence that JF should be further investigated as a lead plant in the drug discovery of new uterine relaxants.

A Close Relationship Between Networks of Interstitial Cells of Cajal and Gastrointestinal Transit In Vivo.

The interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are located in the same area as the myenteric plexus. ICC-MP networks are linked to the generation of electrical pacemaker activity that causes spontaneous gastrointestinal (GI) contractions; however, its role in GI transit is not clear. The aim of this study was to comprehensively investigate the effect of ICC-MP disruption on GI transit in vivo using W/W v mice, partially ICC-deficient model mice. In this study, we measured GI transit using a 13C-octanoic acid breath test, an orally administered dye and a bead expulsion assay. ICC were detected by immunohistochemical staining for c-Kit, a specific marker for ICC. Interestingly, we found that gastric emptying in W/W v mice was normal. We also found that the ability of small intestinal and colonic transit was significantly reduced in W/W v mice. Immunohistochemical staining using whole-mount muscularis samples revealed that c-Kit-positive ICC-MP networks were formed in wild-type mice. In contrast, ICC-MP networks in W/W v mice were maintained only in the gastric antrum and were significantly reduced in the ileum and colon. No significant changes were observed in the nerve structures of the myenteric plexus in W/W v mice. These findings suggest that ICC-MP contribute to GI transit as a powerful driving function in vivo.

Mucosa-Dependent, Stretch-Sensitive Spontaneous Activity in Seminal Vesicle.

Seminal vesicles (SVs), a pair of male accessory glands, contract upon sympathetic nerve excitation during ejaculation while developing spontaneous phasic constrictions in the inter-ejaculatory storage phase. Recently, the fundamental role of the mucosa in generating spontaneous activity in SV of the guinea pig has been revealed. Stretching the mucosa-intact but not mucosa-denuded SV smooth muscle evokes spontaneous phasic contractions arising from action potential firing triggered by electrical slow waves and associated Ca2+ flashes. These spontaneous events primarily depend on sarco-endoplasmic reticulum (SR/ER) Ca2+ handling linked with the opening of Ca2+-activated chloride channels (CaCCs) resulting in the generation of slow waves. Slow waves in mucosa-intact SV smooth muscle are abolished upon blockade of gap junctions, suggesting that seminal smooth muscle cells are driven by cells distributed in the mucosa. In the SV mucosal preparations dissected free from the smooth muscle layer, a population of cells located just beneath the epithelium develop spontaneous Ca2+ transients relying on SR/ER Ca2+ handling. In the lamina propria of the SV mucosa, vimentin-immunoreactive interstitial cells including platelet-derived growth factor receptor α (PDGFRα)-immunoreactive cells are distributed, while known pacemaker cells in other smooth muscle tissues, e.g. c-Kit-positive interstitial cells or α-smooth muscle actin-positive atypical smooth muscle cells, are absent. The spontaneously-active subepithelial cells appear to drive spontaneous activity in SV smooth muscle either by sending depolarizing signals or by releasing humoral substances. Interstitial cells in the lamina propria may act as intermediaries of signal transmission from the subepithelial cells to the smooth muscle cells.

Inhibitory effect of Bisphenol A on in vitro feline uterine contractions.

Bisphenol A (BPA) is an environmental pollutant used as a plasticizer in the manufacture of many plastic products, such as packaging, containers, and water and beverage bottles. There are deleterious effects of BPA on metabolic, endocrine, nervous, and reproductive systems. This is the first study in which there was investigation of the in vitro effect of BPA on the spontaneous contractions of the cat uterus. The tubal uterine segments (1 cm) collected from queens in estrus were suspended in an isolated organ bath. Following tissue stabilization, spontaneous contractions were recorded for 10 min to constitute the control group. The effects of the solvent (alcohol) and BPA at different concentrations (1, 10, and 100 µM) on uterine contractions were subsequently evaluated at 10 min intervals in terms of frequency and mean amplitude variables. The ethanol vehicle did not alter the uterine contractions compared to the control group. All concentrations of BPA used in the study resulted in a reduction (P < 0.05) in amplitude of uterine contractions in a dose-dependent manner, while only the largest dose of BPA decreased the frequency of contractions (P < 0.05). In reproductive physiology, regular uterine contractions facilitate successful fertilization, migration, implantation, and maintenance of pregnancy, as well as fetus expulsion. The results of this study indicate BPA has an inhibitory effect on spontaneous contractions of the cat uterus. It is proposed that this suppressive effect of BPA on uterine contractions might lead to queen infertility.

The role of HCN channels in peristaltic dysfunction in human ureteral tuberculosis.

OBJECTIVE: To explore the role of HCN channels in ureteral peristaltic dysfunction by comparing the changes in HCN channel levels between normal and tuberculous ureters. METHODS: A total of 32 specimens of human upper ureters were collected by nephrectomy from patients with renal tumor (control group, n = 16) or from patients with renal tuberculosis (experimental group, n = 16); the two groups did not receive radiotherapy, chemotherapy, immunotherapy, or any other special treatment before the surgical procedure. An experimental study on smooth muscle strips of human upper ureters showed variation in contraction amplitude and frequency after adding ZD7288, a specific blocker of HCN channels. The expression of HCN channels in the ureter was confirmed by Western blot (WB) and by confocal analysis of double immunostaining for c-kit and HCN channel proteins. RESULTS: Before the addition of ZD7288, the experimental and control groups showed significant differences in the frequency and amplitude of the spontaneous contraction of isolated ureteral smooth muscle strips. After ZD7288 was added, the frequency and amplitude of the contractions of the ureteral smooth muscle strips were significantly lower in both groups. The differences observed before and after ZD7288 treatment in each group were significant (P < 0.001), and the difference in contraction amplitude observed between the two groups before ZD7288 was also significantly different (P < 0.001). By using WB technology, we showed that the expression of HCN channels was present in normal human ureters, with the expression of HCN4 and HCN1 being the highest; the expression of HCN4 and HCN1 in the control and experimental groups were both statistically significant (P < 0.001). HCN4 and HCN1 were expressed in the mucosal and smooth muscle layers of human control ureters and tuberculous ureters, as revealed by a confocal analysis of double immunostaining for c-kit and HCNs proteins; there were significant differences between the two groups (P < 0.001). CONCLUSION: Four HCN channels are expressed in the ureter, mainly HCN4 and HCN1, suggesting that HCN channels are involved in the peristaltic contraction of ureteral ICCs, which may be an important reason for peristaltic dysfunction in ureteric tuberculosis.

Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle.

KEY POINTS: The mucosa may have neuron-like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+ . These spontaneous events primarily depend on Ca2+ handling by sarco-endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco-endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. ABSTRACT: The role of the mucosa in generating the spontaneous activity of guinea-pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi-fluorescence Ca2+ imaging, respectively. Mucosa-intact but not mucosa-denuded SV preparations generated TTX- (1 µm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 µm). Consistently, SMCs developed mucosa-dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 µm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 µm), a sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) inhibitor. DIDS (300 µm) and niflumic acid (100 µm), blockers for Ca2+ -activated Cl- channels (CACCs), or low Cl- solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L-type voltage-dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco-endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.

On the origin of spontaneous activity in the bladder.

OBJECTIVES: To characterise separately the pharmacological profiles of spontaneous contractions from the mucosa and detrusor layers of the bladder wall and to describe the relationship in mucosa between adenosine triphosphate (ATP) release and spontaneous contractions. MATERIALS AND METHODS: Spontaneous contractions were measured (36 °C) from isolated mucosa or detrusor preparations, and intact (mucosa + detrusor) preparations from guinea-pig bladders. Potential modulators were added to the superfusate. The percentage of smooth muscle was measured in haematoxylin and eosin stained sections. ATP release was measured in superfusate samples from a fixed point above the preparation using a luciferin-luciferase assay. RESULTS: The magnitude of spontaneous contractions was in the order intact >mucosa >detrusor. The percentage of smooth muscle was least in mucosa and greatest in detrusor preparations. The pharmacological profiles of spontaneous contractions were different in mucosa and detrusor in response to P2X or P2Y receptor agonists, adenosine and capsaicin. The intact preparations showed responses intermediate to those from mucosa and detrusor preparations. Low extracellular pH generated large changes in detrusor, but not mucosa preparations. The mucosa preparations released ATP in a cyclical manner, followed by variations in spontaneous contractions. ATP release was greater in mucosa compared with detrusor, augmented by carbachol and reversed by the M2 -selective antagonist, methoctramine. CONCLUSIONS: The different pharmacological profiles of bladder mucosa and detrusor, implies different pathways for contractile activation. Also, the intermediate responses from intact preparations implies functional interaction. The temporal relationship between cyclical variation of ATP release and amplitude of spontaneous contractions is consistent with ATP release controlling spontaneous activity. Carbachol-mediated ATP release was independent of active contractile force.

Dexmendetomidine decreases spontaneous contraction of duodenal smooth muscle of rabbits in vitro / 中国药理学通报

Aim To observe the influences of dexmen-detomidine on the spontaneous contraction of duodenal smooth muscle of rabbits in vitro and explore the mech-anisms.Methods The rabbits ( male or female ) were stunned and the duodenums were isolated .The sam-ples of duodenal segments were connected with tension transducer , which were then put into oxygen saturation Krebs-Henseleit ( K-H) solution .The influences of dex-mendetomidine on amplitude ( AM ) and frequency ( FR ) of duodenal smooth muscle were recorded by BL-420 F biological signal processing system .The cu-mulative dosing method was used to observe the differ-ent concentrations of dexmedetomidine on duodenal smooth muscle spontaneous contraction .Glibenclamide ( Gli) was added to K-H solution before dexmendeto-midine.In the calcium-free K-H solution, calcium chloride and rynodine were added before dexmendeto-midine.The mechanisms of dexmendetomidine were studied .Results ① Dexmendetomidine reduced the amplitude of spontaneous contraction of duodenal smooth muscle in rabbits in a dose-dependent manner ( P0.05 ) .② Gli ( P <0.05 ) partly abolished the inhibitory effects of dexmendetomi-dine on duodenal smooth muscle .③ Dexmendetomi-dine inhibited the contraction of duodenum smooth muscle induced by calcium chloride ( P <0.05 ) and rynodine ( P<0.05 ) application into calcium-free K-H solution.Conclusion Dexmendetomidine inhibits the spontaneous contraction of duodenal smooth muscle of rabbits in vitro.The mechanisms may be related to ac-tivating ATP sensitive potassium channels , inhibition of the extracellular calcium influx via cell membrane and intracellular calcium release via sarcoplasmic reticulum in duodenal smooth muscle .

Effect of gingerol on colonic motility via inhibition of calcium channel currents in rats.

AIM: To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process. METHODS: The distal colon was cut along the mesenteric border and cleaned with Ca(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique. RESULTS: Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 µmol/L, 50 µmol/L, 75 µmol/L and 100 µmol/L, respectively (P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 µmol/L concentration of gingerol, the percentage of gingerol-induced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6% (P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 mV, respectively, at concentrations of 25 µmol/L, 50 µmol/L, 75 µmol/L and 100 µmol/L (P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was -14.23 ± 1.12 mV in the control group and -10.56 ± 1.04 mV in the 75 µmol/L group (P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93 (P < 0.05) in the control and 75 µmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of -27.43 ± 1.26 mV in the control group and -26.56 ± 1.53 mV in the 75 µmol/L gingerol group (P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68 (P > 0.05) in the 75 µmol/L gingerol group. CONCLUSION: Gingerol inhibits colonic motility by preventing Ca(2+) influx through L-type calcium channels.

Role of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in spontaneous contrac-tion of smooth muscles in human isolated ureters / 局解手术学杂志

Objective To investigate effects of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in the hu-man ureter on the spontaneous contraction of smooth muscles. Methods Four HCN subtypes were detected in human ureteral tissue using reverse transcription polymerase chain reaction,Western blotting and immunohistochemical. ZD7288,the HCN blocker, was used to observe the changes of ureteral muscle contraction amplitude and frequency by applying the ureteral smooth muscle strip test in vitro. Results HCN1-4 isoforms were all identified in human ureter using reverse transcription-polymerase chain reaction and Western blotting. Through the immunohistochemical,HCN channel was found mostly in the urothelium layer and muscular layer of human ureteral wall. ZD7288 significantly decreased the bladder excitation. Conclusion All 4 HCN channel hypotypes exist in the human ureter, and affect the ureteral excitation.

Relaxant effect of flavonoid naringenin on contractile activity of rat colonic smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Disturbed gastrointestinal (GI) motility can be associated with smooth muscle abnormalities and dysfunction. Exploring innovative approaches that can modulate the disturbed colonic motility are of great importance for clinical therapeutics. Naringenin, a flavonoid presented in many traditional Chinese herbal medicines, has been shown to have a relaxant effect on different smooth muscles. The aim of the present study was to investigate the effect of naringenin on regulation of GI motility. MATERIAL AND METHODS: Mechanical recording was used to investigate the effect of naringenin on isolated rat colonic smooth muscle spontaneous contractions. Whole cell patch clamp, intracellular [Ca(2+)] concentration ([Ca(2+)]i) and membrane potential measurements were examined on primary cultures of colonic smooth muscle cells (SMCs). A neostigmine-stimulated rat model was utilized to investigate the effect of naringenin in vivo. RESULTS: Naringenin induced a concentration-dependent inhibition (1-1000 µM) on rat colonic spontaneous contraction, which was reversible after wash out. The external Ca(2+) influx induced contraction and [Ca(2+)]i increase were inhibited by naringenin (100 µM). In rat colonic SMCs, naringenin-induced membrane potential hyperpolarization was sensitive to TEA and selective large-conductance calcium-activated K(+) (BKCa) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, naringenin stimulated an iberiotoxin-sensitive BKCa current, which was insensitive to changes in the [Ca(2+)]i concentration. Furthermore, naringenin significantly suppressed neostigmine-enhanced rat colon transit in vivo. CONCLUSION: Our results for the first time demonstrated the relaxant effect of flavonoid naringenin on colon smooth muscle both in vitro and in vivo. The relaxant effect of naringenin was attributed to direct activation of BKCa channels, which subsequently hyperpolarized the colonic SMCs and decreased Ca(2+) influx through VDCC. Naringenin might be of therapeutic value in the treatment of GI motility disorders.

Effects of Rho-kinase inhibition on myosin light chain phosphorylation and obstruction-induced detrusor overactivity.

OBJECTIVES: To study the relationship between myosin light chain phosphorylation of the detrusor muscle and spontaneous smooth muscle contractions in a rabbit model of partial outlet obstruction. METHODS: New Zealand white rabbit urinary bladders were partially obstructed for 2 weeks. Rabbits were euthanized, detrusor muscle strips were hung on a force transducer and spontaneous activity was measured at varying concentrations (0-0.03 µM/L) of the Rho-kinase inhibitors GSK 576371 or 0.01 µM/L Y27632. Basal myosin light chain phosphorylation was measured by 2-D gel electrophoresis in control and GSK 576371-treated strips. RESULTS: Both drugs suppressed the force of spontaneous contractions, whereas GSK 576371 had a more profound effect on the frequency of the contractions. The IC50 values for the inhibition of frequency and force of spontaneous contractions were 0.17 µM/L and 0.023 µM/L for GSK 576371, respectively. The compound significantly decreased the basal myosin light chain phosphorylation from 28.0 ± 3.9% to 13.5 ± 1.9% (P < 0.05). At 0.01 µM/L, GSK 576371 inhibited spontaneous bladder overactivity by 50%, but inhibited carbachol-elicited contractions force by just 25%. CONCLUSIONS: These data suggest that Rho-kinase regulation of myosin light chain phosphorylation contributes to the spontaneous detrusor activity induced by obstruction. This finding could have therapeutic implications by providing another therapeutic option for myogenic, overactive bladder.

Maximum shortening velocity of lymphatic muscle approaches that of striated muscle.

Lymphatic muscle (LM) is widely considered to be a type of vascular smooth muscle, even though LM cells uniquely express contractile proteins from both smooth muscle and cardiac muscle. We tested the hypothesis that LM exhibits an unloaded maximum shortening velocity (Vmax) intermediate between that of smooth muscle and cardiac muscle. Single lymphatic vessels were dissected from the rat mesentery, mounted in a servo-controlled wire myograph, and subjected to isotonic quick release protocols during spontaneous or agonist-evoked contractions. After maximal activation, isotonic quick releases were performed at both the peak and plateau phases of contraction. Vmax was 0.48 ± 0.04 lengths (L)/s at the peak: 2.3 times higher than that of mesenteric arteries and 11.4 times higher than mesenteric veins. In cannulated, pressurized lymphatic vessels, shortening velocity was determined from the maximal rate of constriction [rate of change in internal diameter (-dD/dt)] during spontaneous contractions at optimal preload and minimal afterload; peak -dD/dt exceeded that obtained during any of the isotonic quick release protocols (2.14 ± 0.30 L/s). Peak -dD/dt declined with pressure elevation or activation using substance P. Thus, isotonic methods yielded Vmax values for LM in the mid to high end (0.48 L/s) of those the recorded for phasic smooth muscle (0.05-0.5 L/s), whereas isobaric measurements yielded values (>2.0 L/s) that overlapped the midrange of values for cardiac muscle (0.6-3.3 L/s). Our results challenge the dogma that LM is classical vascular smooth muscle, and its unusually high Vmax is consistent with the expression of cardiac muscle contractile proteins in the lymphatic vessel wall.

Effect of the ginsenoside Rb1 on the spontaneous contraction of intestinal smooth muscle in mice.

AIM: To investigate the effect and the possible mechanism of ginsenoside Rb1 on small intestinal smooth muscle motility in mice. METHODS: Intestinal smooth muscle strips were isolated from male ICR mice (5 wk old), and the effect of ginsenoside Rb1 on spontaneous contraction was recorded with an electrophysiolograph. The effect of ginsenoside Rb1 on ion channel currents, including the voltage-gated K⁺ channel current (IK(V)), calcium-activated potassium channel currents (IK(Ca)), spontaneous transient outward currents and ATP-sensitive potassium channel current (IK(ATP)), was recorded on freshly isolated single cells using the whole-cell patch clamp technique. RESULTS: Ginsenoside Rb1 dose-dependently inhibited the spontaneous contraction of intestinal smooth muscle by 21.15% ± 3.31%, 42.03% ± 8.23% and 67.23% ± 5.63% at concentrations of 25 µmol/L, 50 µmol/L and 100 µmol/L, respectively (n = 5, P < 0.05). The inhibitory effect of ginsenoside Rb1 on spontaneous contraction was significantly but incompletely blocked by 10 mmol/L tetraethylammonium or 0.5 mmol/L 4-aminopyridine, respectively (n = 5, P < 0.05). However, the inhibitory effect of ginsenoside Rb1 on spontaneous contraction was not affected by 10 µmol/L glibenclamide or 0.4 µmol/L tetrodotoxin. At the cell level, ginsenoside Rb1 increased outward potassium currents, and IK(V) was enhanced from 1137.71 ± 171.62 pA to 1449.73 ± 162.39 pA by 50 µmol/L Rb1 at +60 mV (n = 6, P < 0.05). Ginsenoside Rb1 increased IK(Ca) and enhanced the amplitudes of spontaneous transient outward currents from 582.77 ± 179.09 mV to 788.12 ± 278.34 mV (n = 5, P < 0.05). However, ginsenoside Rb1 (50 µmol/L) had no significant effect on IK(ATP) (n = 3, P < 0.05). CONCLUSION: These results suggest that ginsenoside Rb1 has an inhibitory effect on the spontaneous contraction of mouse intestinal smooth muscle mediated by the activation of IK(V) and IK(Ca), but the K(ATP) channel was not involved in this effect.

Spontaneous Contractile Activity of the Detrusor Muscle and Its Role in the Pathogenesis of Overactive Bladder Syndrome.

There is accumulated evidence that spontaneous contractions (SCs) in the bladder wall are associated with afferent nerve firing in the bladder. The role of the urothelium in bladder sensation might be restricted to pathological conditions, such as interstitial cystitis or chemical cystitis in which the release of urothelium-derived mediators such as adenosine triphosphate is increased. Recent publications imply that SCs in bladders with detrusor overactivity due to spinal cord injury or bladder outlet obstruction are modulated by intracellular signal transduction mechanisms such as the RhoA/Rho-kinase pathway, denervation-supersensitivity to acetylcholine, changes in ion channel activity, enhanced gap-junctional intercellular communication, alterations in interstitial cells of Cajal, the actions of local mediators in the detrusor and the influence of the urothelium. Spontaneous contractions and possible consequent afferent nerve firing might participate in the generation of overactive bladder syndrome.