Acetylcholine exerts inhibitory and excitatory actions on mouse ileal pacemaker activity: role of muscarinic versus nicotinic receptors.

The effect of acetylcholine (ACh) on pacemaking and spontaneous contractions in the gastrointestinal tract is not well characterized. The current study aims to profile the effect of several muscarinic and nicotinic receptor agonists and antagonists on pacemaker potentials in the ICR mouse ileum. Pacemaker potentials of whole thickness mouse ileal segments were recorded extracellularly using a 60-channel microelectrode array (MEA) platform. A spatiotemporal analysis integrated the frequency, amplitude, and velocity measurements of pacemaker currents. Comparative data were obtained by recording spontaneous smooth muscle tone in a conventional organ bath. On the MEA, ACh (0.3-300 µM) and bethanechol (0.3-300 µM) significantly reduced ileal pacemaker potentials. The inhibitory effect of ACh was mimicked by donepezil (300 µM) but not nicotine (0.3-7 mM). Atropine (300 µM), but not hexamethonium (300 µM), reversed the inhibitory actions of ACh and bethanechol and revealed excitatory properties manifested as increases in pacemaker frequency. A spatial analysis also revealed that atropine, but not hexamethonium, reversed the ACh-induced distortion of pacemaker propagation activity. Atropine (0.001-3 mM) and hexamethonium (0.3-7 mM) alone were inactive. In the organ bath, ACh (300 nM) and bethanechol (30 µM) induced ileal tonic contractions, while inhibiting basal spontaneous contractions at 300 µM. Atropine (1 µM), but not hexamethonium (1-300 µM), reversed both the tonic contractions and the inhibition of the spontaneous contractions of ACh and bethanechol and revealed an excitatory effect manifested as an increasing in the frequency of contractions. Muscarinic, but not nicotinic, receptors appear to mediate the inhibitory actions of ACh on mouse ileal pacemaker potentials.NEW & NOTEWORTHY The study discovered an acute action of acetylcholine on pacemaker potentials that is mediated by muscarinic receptors on the mouse ileum. Bethanechol, but not nicotine, mimicked the inhibitory actions of acetylcholine on pacemaker potentials. Atropine, but not hexamethonium, reversed the inhibitory actions of acetylcholine. When introduced after acetylcholine, atropine exhibited excitatory actions that increased the pacemaker frequency. Acetylcholine and bethanechol distorted the propagation activity and pattern, and this was also reversed by atropine. These actions of acetylcholine on pacemaker potentials may contribute to pathophysiology in bowel diseases.

Author Correction: TRPC5 channels participate in pressure-sensing in aortic baroreceptors.

This corrects the article DOI: 10.1038/ncomms11947.

TRPC5 channels participate in pressure-sensing in aortic baroreceptors.

Blood pressure is maintained within a normal physiological range by a sophisticated regulatory mechanism. Baroreceptors serve as a frontline sensor to detect the change in blood pressure. Nerve signals are then sent to the cardiovascular control centre in the brain in order to stimulate baroreflex responses. Here, we identify TRPC5 channels as a mechanical sensor in aortic baroreceptors. In Trpc5 knockout mice, the pressure-induced action potential firings in the afferent nerve and the baroreflex-mediated heart rate reduction are attenuated. Telemetric measurements of blood pressure demonstrate that Trpc5 knockout mice display severe daily blood pressure fluctuation. Our results suggest that TRPC5 channels represent a key pressure transducer in the baroreceptors and play an important role in maintaining blood pressure stability. Because baroreceptor dysfunction contributes to a variety of cardiovascular diseases including hypertension, heart failure and myocardial infarction, our findings may have important future clinical implications.

Antitumor effects of novel compound, guttiferone K, on colon cancer by p21Waf1/Cip1-mediated G(0) /G(1) cell cycle arrest and apoptosis.

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration- and time-dependently reduced the viability of human colon cancer HT-29 cells (IC(50) value 5.39 ± 0.22 µM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G(0) /G(1) cell cycle arrest in HT-29 cells by down-regulating cyclins D1, D3 and cyclin-dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 µM) induced cleavage of PARP, caspases-3, -8 and -9 and chromatin condensation to stimulate caspase-3-mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK-induced caspase-3 activity, indicating the possible involvement of JNK in GUTK-induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5-fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase-3 and a decrease in cell proliferation marker Ki-67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.

The use of SU-8 topographically guided microelectrode array in measuring extracellular field potential propagation.

The microelectrode array (MEA) can be used to study extracellular field potentials (exFPs) of electrogenic cells. Microcontact printing, which must be repeated after each experiment, is often used to promote accurate positioning of cells onto electrodes. The present study used MEAs with evenly spaced detection electrodes aligning along permanent SU-8 topographical guidance channels to measure propagation direction and speed. Chronotropic agents, isoproterenol (ISO, 1 nM-1 mM), and verapamil (VP, 1 nM-10 µM); and potassium channel openers (KCOs), pinacidil (PIN), and SDZ PCO400 (SDZ), were used to characterize these MEA chips. ISO (1 mM) enhanced the propagation speed from 247.25 ± 50.58 µm/ms 381.29 ± 92.01 µm/ms (n = 9, p < 0.05), whereas VP (10 µM) reduced the propagation speed completely (n = 12, p < 0.001). PIN (1 mM) significantly reduced the propagation speed from 278.6 ± 43.7 µm/ms to 49.7 ± 27.7 µm/ms (n = 10, p < 0.001), whereas SDZ (1 mM) completely stopped the propagation (n = 9, p < 0.001). Both KCOs induced conduction pattern changes similar to those observed in cardiac arrhythmia. The MEA chips with SU-8 guidance channels may be used to study cardiovascular diseases that are related to conduction disruption.

A physiological role of glucagon-like peptide-1 receptors in the central nervous system of Suncus murinus (house musk shrew).

Glucagon-like peptide-1 (7-36) amide (GLP-1) is released from the gut as an incretin hormone to stimulate glucose-stimulated insulin secretion. GLP-1 is also produced in the central nervous system (CNS) as a neurotransmitter that regulates feeding behaviour. By using polyclonal antiserum against GLP-1 and GLP-1 receptors, we identified the distribution of GLP-1 immunoreactive fibres and GLP-1 receptor immunoreactivity in the ventromedial hypothalamus of Suncus murinus (house musk shrew). In functional studies, subcutaneous administration of exendin-4 (1 - 30 nmol/kg) reduced blood glucose levels dose-dependently by up to 49% during an intraperitoneal glucose tolerance test (P<0.001). The glucose-lowering effects were also observed after an intracerebroventricular (i.c.v.; 0.3 - 3 nmol) or intracerebral ventromedial hypothalamic microinfusion (iVMH; 0.3 - 3 pmol) of exendin-4. The area under the curve values for glucose after i.c.v. and iVMH administrations of exendin-4 were reduced by up to 53% (P<0.01) and 46% (P<0.01), respectively. Exendin-4 (i.c.v.; 3 nmol) also increased glucose-stimulated insulin secretion by 20% compared to controls (P<0.05). The GLP-1 receptor antagonist, exendin (9-39) (10 nmol, i.c.v.) did not modify blood glucose levels but it antagonized the glucose-lowering effect of exendin-4 (1 nmol, i.c.v.; P<0.05). The data suggests that the central GLP-1 system may regulate glucose homeostasis by increasing insulin secretion. Further, GLP-1 receptors in the ventromedial hypothalamus appear to play an important role in the regulation of glucose homeostasis in S. murinus.

A study of the relationship between pharmacologic preconditioning and adenosine triphosphate-sensitive potassium (KATP) channels on cultured cardiomyocytes using the microelectrode array.

The microelectrode array was used to study the pharmacologic preconditioning effect of adenosine triphosphate-sensitive channel activation using potassium channel openers (KCOs) on rat cardiomyocytes over 90 minutes of ischemia. Cell viability and electrophysiological changes between KCOs pretreated and untreated cardiomyocytes were compared. Ischemia caused significant increases in beat frequency, extracellular field potential amplitude, and propagation velocity of spontaneously beating untreated cardiomyocytes. However, these electrophysiological parameters reduced as the duration of ischemia increased. The electrophysiological changes on ischemic cardiomyocytes were abolished by pretreating the cells with KCOs. Pinacidil pretreated cardiomyocytes retained a significantly higher viability than the untreated cardiomyocytes after 90 minutes of ischemia. Because Connexin 43 has a direct correlation with the propagation velocity, the Connexin 43 protein expression was also investigated. Connexin 43 expression levels were lower in KCOs pretreated cardiomyocytes than that of the untreated controls, and this correlated with the propagation velocity results obtained from the microelectrode array. The effect of pinacidil (sarcolemmal adenosine triphosphate-sensitive channel opener) was more prominent than that of diazoxide (mitochondrial K adenosine triphosphate-sensitive channel opener) on ischemic cardiomyocytes as indicated in the present acute ischemia study.

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection.

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 degrees C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and gamma-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.

Modulatory action of potassium channel openers on field potential and histamine release from rat peritoneal mast cells.

To determine whether changes in membrane potential affect the extent of mast cell degranulation, compound 48/80 was added to rat peritoneal mast cell suspensions in the absence or presence of potassium channel openers (KCOs). Changes were compared between the field potential (FP) and the amount of histamine released. The results demonstrated that (i) the onset and duration of FP, which reflects the hyperpolarizing nature of the response, increased as the concentration of compound 48/80 increased; (ii) both FP and the amount of histamine released increased as the concentration of compound 48/80 increased; (iii) although both KCOs (SDZ PCO400, a benzopyran derivative, and P1060, a cyanoguanidine derivative) potentiated compound 48/80-induced increases in FP and histamine release, without compound 48/80, they had no effect on either parameter; (iv) both glibenclamide and charybdotoxin significantly attenuated the compound 48/80-induced increase in FP; and (v) glibenclamide was able to attenuate the KCO-induced potentiation of FP. The results show that drugs presumably causing hyperpolarization can affect histamine release from rat peritoneal mast cells. The effect of KCOs on compound 48/80-induced response appears to be potentiation in nature rather than synergism. It is possible that KCO hyperpolarizes the cell membrane, enhances Ca2+ influx, and thus increases histamine release. As such, selective blockers of K+ channels may be useful for the treatment of immunological disorders.

To establish a pharmacological experimental platform for the study of cardiac hypoxia using the microelectrode array.

INTRODUCTION: Simultaneous recording of electrical potentials from multiple cells may be useful for physiological and pharmacological research. The present study aimed to establish an in vitro cardiac hypoxia experimental platform on the microelectrode array (MEA). METHODS: Embryonic rat cardiac myocytes were cultured on the MEAs. Following >or=90 min of hypoxia, changes in lactate production (mM), pH, beat frequency (beats per min, bpm), extracellular action potential (exAP) amplitude, and propagation velocity between the normoxic and hypoxic cells were compared. RESULTS: Under hypoxia, the beat frequency of cells increased and peaked at around 42.5 min (08.1+/-3.2 bpm). The exAP amplitude reduced as soon as the cells were exposed to the hypoxic medium, and this reduction increased significantly after approximately 20 min of hypoxia. The propagation velocity of the hypoxic cells was significantly lower than that of the control throughout the entire 90+ min of hypoxia. The rate of depolarisation and Na(+) signal gradually reduced over time, and these changes had a direct effect on the exAP duration. DISCUSSION: The extracellular electrophysiological measurements allow a partial reconstruction of the signal shape and time course of the underlying hypoxia-associated physiological changes. The present study showed that the cardiac myocyte-integrated MEA may be used as an experimental platform for the pharmacological studies of cardiovascular diseases in the future.

The use of microelectrode array (MEA) to study rat peritoneal mast cell activation.

We performed this study to demonstrate the applicability of the microelectrode array (MEA) to study electrophysiological changes of rat peritoneal mast cells in the presence of compound 48/80 under normal, Ca(2+)-free, Ca(2+)-free with EDTA, and Cl(-)-free conditions. The use of high extracellular K(+) (KCl, 150 mM), charybdotoxin (ChTX, 100 nM), and Cl(-)-free containing ChTX buffers verified that the hyperpolarizing signal was due to the activation of mainly K(+) and, to a lesser extent, Cl(-) channels. Compound 48/80 concentration-dependently shortened the latent periods (the onset of response) and increased both the spatial (the K(+) and Cl(-) hyperpolarizing field potentials, HFP) and temporal measurements (the duration of response). Ca(2+)-free buffer had no effect on the latent period of compound 48/80 but increased the HFP at high concentrations. The latent period increased while the HFP diminished when cells were equilibrated in Ca(2+)-free buffer containing EDTA. Durations of the HFP were generally longer when cells were in either Ca(2+)-free or Ca(2+)-free containing EDTA buffers than when cells were in normal buffer. The EC(50) values confirmed that effects were only affected in Ca(2+)-free buffer containing EDTA but not in Ca(2+)-free or Cl(-)-free buffers, further reinforcing the hypothesis that the presence of Ca(2+) is not essential to the action of compound 48/80. The present study is the first application of MEA to study rat peritoneal mast cells, and our results indicate that it could be of value in future pharmacological research on other non-excitable cells.