A highly selective bulk optode based on 6-{4-(2,4-dihydroxy-phenyl)diazenyl)phenyl}-2-oxo-4-phenyl-1,2-dihydro-pyridine-3-carbonitrile incorporating chromoionophore V for determination of nano levels of cadmium.

A novel optical sensor has been fabricated for highly accurate, simple and selective determination of nanomolar levels of cadmium ions. The sensor depends on the interaction of 6-{4-(2,4-dihydroxyphenyl)diazenyl)phenyl}-2-oxo-4-phenyl-1,2-dihydropyri-dine-3-carbonitrile (DDPODC) with Cd(II) in plasticized (2-nitrophenyloctyl ether) (o-NPOE) polyvinylchloride (PVC) membrane incorporating chromoionophore V as a lipophilic H+-selective indicator. It would seem that the higher Cd(II) concentration, the lower absorbance of chromoionophore V in the membrane at 668 nm, whereas the absorbance at 586 nm increased. The developed sensor at pH 4.7 has a linear range of 5.0 × 10-12 - 2.5 × 10-5 M with limits of detection and quantification of 1.62 × 10-12 and 4.95 × 10-12 M, respectively. The relative standard deviation (RSD) for eight determination of 1.0 × 10-7 M Cd(II) was 1.67%. Finally, the proposed sensor gives good results for applications in the direct determination of cadmium ions in water, food, and biological samples. Additionally, we compared the obtained results with the data obtained from the flame atomic absorption spectrometry (FAAS).

2-Amino-4-aryl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitriles with Microtubule-Disruptive, Centrosome-Declustering, and Antiangiogenic Effects in†vitro and in†vivo.

A series of fifteen 2-amino-4-aryl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitriles (1 a-o) were synthesized via a three-component reaction of 4-hydroxycoumarin, malononitrile, and diversely substituted benzaldehydes or pyridine carbaldehydes. The compounds were tested for anticancer activities against a panel of eight human tumor cell lines. A few derivatives with high antiproliferative activities and different cancer cell specificity were identified and investigated for their modes of action. They led to microtubule disruption, centrosome de-clustering and G2/M cell cycle arrest in 518 A2 melanoma cells. They also showed anti-angiogenic effects in vitro and in vivo.

Role of prediction error and the cholinergic system on memory reconsolidation processes in mice.

The ability to make predictions based on stored information is a general coding strategy. A prediction error (PE) is a mismatch between expected and current events. Our memories, like ourselves, are subject to change. Thus, an acquired memory can become active and update its content or strength by a labilization-reconsolidation process. Within the reconsolidation framework, PE drives the updating of consolidated memories. In the past our lab has made key progresses showing that a blockade in the central cholinergic system during reconsolidation can cause memory impairment, while reinforcement of cholinergic activity enhances it. In the present work we determined that PE is a necessary condition for memory to reconsolidate in an inhibitory avoidance task using both male and female mice. Depending on the intensity of the unconditioned stimulus (US) used during training, a negative (higher US intensity) or positive (lower US intensity/no US) PE on a retrieval session modified the behavioral response on a subsequent testing session. Furthermore, we demonstrated that the cholinergic system modulates memory reconsolidation only when PE is detected. In this scenario administration of oxotremorine, scopolamine or nicotine after memory reactivation either enhanced or impaired memory reconsolidation in a sex-specific manner.

SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic ß cells.

SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of ß-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic ß cells.

Acetylcholine-dependent upregulation of TASK-1 channels in thalamic interneurons by a smooth muscle-like signalling pathway.

KEY POINTS: The ascending brainstem transmitter acetylcholine depolarizes thalamocortical relay neurons while it induces hyperpolarization in local circuit inhibitory interneurons. Sustained K+ currents are modulated in thalamic neurons to control their activity modes; for the interneurons the molecular nature of the underlying ion channels is as yet unknown. Activation of TASK-1 K+ channels results in hyperpolarization of interneurons and suppression of their action potential firing. The modulation cascade involves a non-receptor tyrosine kinase, c-Src. The present study identifies a novel pathway for the activation of TASK-1 channels in CNS neurons that resembles cholinergic signalling and TASK-1 current modulation during hypoxia in smooth muscle cells. ABSTRACT: The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay neurons by inhibiting two-pore domain potassium (K2P ) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors induces IN hyperpolarization by recruiting the G-protein ßγ subunit (Gßγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase, and cellular and sarcoma (c-Src) tyrosine kinase, leading to activation of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK)-1 channels. The latter was confirmed by the use of TASK-1-deficient mice. Furthermore inhibition of phospholipase Cß as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src tyrosine kinase in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons.

Photouncaged Sequence-specific Interstrand DNA Cross-Linking with Photolabile 4-oxo-enal-modified Oligonucleotides.

DNA cross-linking technology is an attractive tool for the detection, regulation, and manipulation of genes. In this study, a series of photolabile 4-oxo-enal-modified oligonucleotides functionalized with photosensitive ο-nitrobenzyl derivatives were rationally designed as a new kind of photocaged cross-linking agents. A comprehensive evaluation of cross-linking reactions for different nucleobases in complementary strands under different conditions suggested that the modified DNA oligonucleotides tended to form interstrand cross-linking to nucleobases with the potential of thymidine > guanosine ¼ cytidine ~ adenosine. Different from previous literature reports that cytidine and adenosine were preferential cross-linked nucleobases with 4-oxo-enal moieties, our study represents the first example of DNA cross-linking for T and G selectivity using 4-oxo-enal moiety. The cross-linked adducts were identified and their cross-linking mechanism was also illustrated. This greatly expands the applications of 4-oxo-enal derivatives in the studies of DNA damage and RNA structure.

The effect of dorsal hippocampal administration of nicotinic and muscarinic cholinergic ligands on pentylenetetrazol-induced generalized seizures in rats.

In the present study, the effects of intrahippocampal injections of cholinergic ligands on pentylenetetrazol (PTZ)-induced seizures were investigated in rats. The rats were assigned to 1 of the following 9 groups: saline, nicotine (0.5 or 1 µg), atropine (0.25 or 1 µg), oxotremorine-M (0.1 or 1 µg), or mecamylamine (2 or 8 µg). Cholinergic ligands were administered via intrahippocampal infusion 30 min before seizure induction (intraperitoneal injection of 80 mg/kg PTZ). Results show that antagonists caused nonsignificant increases in the latency of tonic-clonic seizures, significant decreases in the duration of tonic-clonic seizures, significant decreases in the latency of death, and increases in mortality rate. Agonists led to increases in the duration of tonic-clonic seizures, decreases in the latency of death, and decreases in mortality rate. These results provide compelling evidence that cholinergic ligands show modulatory effects on a PTZ model of acute seizure in the rat hippocampus.

Type I interferons impair BDNF-induced cell signaling and neurotrophic activity in differentiated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons.

Type I interferons (IFNs) have been shown to act on neurons and to cause neuronal damage through mechanisms not completely defined. Here, we investigated the effects of type I IFNs on brain-derived neurotrophic factor (BDNF)-induced TrkB receptor signaling and neurotrophic activity. In retinoic acid-treated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons, long-term exposure to IFNs curtailed BDNF-induced activation of phosphatidylinositol 3-kinase, phospholipase Cγ and extracellular-regulated kinases 1 and 2 signaling. Moreover, IFN-ß inhibited BDNF-induced cell survival, neurite outgrowth, and expression of neuronal markers, such as neurofilament proteins, growth-associated protein-43 and glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR1. The IFN inhibitory effects were associated with down-regulation of TrkB and inhibition of TrkB autophosphorylation. In SH-SY5Y cells, blockade of either Janus kinase with pyridone 6 or signal transducer and activator of transcription (STAT) 1 with siRNA transfection attenuated IFN-ß-induced TrkB down-regulation. Quantitative real time RT-PCR indicated that IFN-ß significantly reduced TrkB mRNA levels. Moreover, blockade of protein kinase R counteracted IFN-ß-induced inhibition of TrkB expression and signaling. These data indicate that in neuronal cells IFNs negatively regulate BDNF signaling and neurotrophic activity through inhibition of TrkB activation and Janus kinase/Signal transducer and activator of transcription-dependent down-regulation of TrkB.

Role of M2 and M3 muscarinic acetylcholine receptor subtypes in activation of bladder afferent pathways in spinal cord injured rats.

OBJECTIVE: To evaluate the role of M2 and M3 muscarinic acetylcholine receptor (mAChR) subtypes in the activation of bladder afferent pathways in rats with chronic spinal cord injury (SCI). METHODS: Adult female Sprague-Dawley rats were spinalized at the T9 level. Continuous cystometry was performed under awake conditions 2 or 4 weeks after SCI. The effects of intravesical administration of an mAChR agonist (oxotremorine-methiodide), a nonselective antagonist (atropine), an M2-selective antagonist (methoctramine), and an M3-selective antagonist (darifenacin) were examined. After cystometry, the bladder was removed and separated into the mucosa and detrusor, and the M2 and M3 mAChR mRNA expression in the mucosa was determined using real-time quantitative polymerase chain reaction. RESULTS: At 2 and 4 weeks after SCI, intravesical administration of a nonselective mAChR agonist (25 µM oxotremorine-methiodide) increased the area under the curve of nonvoiding contractions, although the intercontraction interval of voiding contractions and maximal voiding pressure did not change. This effect was blocked by atropine and methoctramine (10 µM) but not by darifenacin (50 µM). However, mAChR antagonists alone (10-50 µM) had no effect on cystometric parameters. M2 mAChR mRNA expression was increased in the mucosa of SCI rats compared with that in normal rats. CONCLUSION: Our results suggest that the M2 mAChR subtype plays an important role in bladder afferent activation that enhances detrusor overactivity in SCI rats. However, because mAChR antagonists alone did not affect any cystometric parameters, the muscarinic mechanism controlling bladder afferent activity might not be involved in the emergence of detrusor overactivity in SCI.

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.

Effects of stimulation of muscarinic receptors on bladder afferent nerves in the in vitro bladder-pelvic afferent nerve preparation of the rat.

Effects of a muscarinic receptor agonist oxotremorine-M (oxo-M) on bladder afferent nerve (BAN) activity were studied in an in vitro bladder-pelvic nerve preparation. Distension of the bladder induced rhythmic bladder contractions that were accompanied by multiunit afferent firing. Intravesical administration of 25 and 50 µM oxo-M significantly increased afferent firing from 41 ± 2 spikes/s to 51 ± 4 spikes/s and 60.5 ± 5 spikes/s, respectively, but did not change the maximum amplitude of spontaneous bladder contractions. The afferent nerve firing induced by isotonic distension of the bladder (10-40 cmH(2)O) was increased 22-100% by intravesical administration of 50 µM oxo-M. Electrical stimulation on the surface of the bladder elicited action potentials (AP) in BAN. Oxo-M significantly decreased the voltage threshold by 40% (p<0.05) and increased by 157% (p<0.05) the area of the AP evoked at a submaximal stimulus intensity. These effects were blocked by intravesical injection of 5 µM atropine methyl nitrate (AMN). Intravesical administration of 5 µM AMN alone did not alter BAN firing or the amplitude of bladder contractions. The facilitatory effects induced by oxo-M on BAN activity were also suppressed (p<0.05) by intravesical administration of 2',3'-0-trinitrophenyl-ATP (TNP-ATP) (30 µM). In preparations pretreated with capsaicin (125 mg/kg, s.c.) the facilitatory effects of 50 µM oxo-M on BAN activity were absent. These results suggest that activation of muscarinic receptors facilitates mechano-sensitive, capsaicin-sensitive BAN activity in part by mechanisms involving purinergic receptors located near the luminal surface of the bladder and ATP release which presumably occurs in the urothelium.

Muscarinic receptor stimulation of D-aspartate uptake into human SH-SY5Y neuroblastoma cells is attenuated by hypoosmolarity.

In addition to its function as an excitatory neurotransmitter, glutamate plays a major role as an osmolyte within the central nervous system (CNS). Accordingly, mechanisms that regulate glutamate release and uptake are of physiological importance not only during conditions in which cell volume remains constant but also when cells are subjected to hypoosmotic stress. In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as D-aspartate) into human SH-SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250-300% of control) in the uptake of D-aspartate and, concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. mAChR-mediated increases in d-aspartate uptake were potently blocked by the EAAT3 inhibitor l-beta-threo-benzyl-aspartate. In hypotonic media, the ability of mAChR activation to facilitate D-aspartate uptake was significantly attenuated (40-50%), and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR-stimulated D-aspartate uptake under hypoosmotic conditions could be fully reversed upon re-exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR-mediated increases in D-aspartate uptake depended on cytoskeletal integrity, protein kinase C and phosphatidylinositol 3-kinase activities, and the availability of intracellular Ca2+. In contrast, dependence on extracellular Ca2+ was observed only under isotonic conditions. The results suggest that, although the uptake of D-aspartate into SH-SY5Y cells is enhanced after mAChR activation, this process is markedly attenuated by hypoosmolarity.

Multiple efflux pumps are involved in the transepithelial transport of colchicine: combined effect of p-glycoprotein and multidrug resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire small intestine.

The purpose of this study was to thoroughly characterize the efflux transporters involved in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine and to evaluate the role of these transporters in limiting its oral absorption. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on colchicine bidirectional permeability were studied across Caco-2 cell monolayers, inhibiting one versus multiple transporters simultaneously. Colchicine permeability was then investigated in different regions of the rat small intestine by in situ single-pass perfusion. Correlation with the P-gp/MRP2 expression level throughout different intestinal segments was investigated by immunoblotting. P-gp inhibitors [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), verapamil, and quinidine], and MRP2 inhibitors [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), indomethacin, and p-aminohippuric acid (p-AH)] significantly increased apical (AP)-basolateral (BL) and decreased BL-AP Caco-2 transport in a concentration-dependent manner. No effect was obtained by the BCRP inhibitors fumitremorgin C (FTC) and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced colchicine mucosal secretion, including complete abolishment of efflux (GF120918/MK571). Colchicine displayed low (versus metoprolol) and constant permeability along the rat small-intestine. GF120918 significantly increased colchicine permeability in the ileum with no effect in the jejunum, whereas MK571 augmented jejunal permeability without changing the ileal transport. The GF120918/MK571 combination caused an effect similar to that of MK571 alone in the jejunum and to that of GF120918 alone in the ileum. P-gp expression followed a gradient increasing from proximal to distal segments, whereas MRP2 decreased from proximal to distal small intestinal regions. Overall, it was revealed that the combined effect of P-gp and MRP2, but not BCRP, dominates colchicine transepithelial transport, leading to complete coverage of the entire small intestine, and makes the efflux transport dominate the intestinal permeability process.

Effects of cholinesterase inhibition in supraspinal and spinal neural pathways on the micturition reflex in rats.

OBJECTIVE: To investigate whether activation of brain and spinal cholinergic pathways affects the micturition reflex in rats. MATERIALS AND METHODS: The effects of intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of neostigmine as a cholinesterase inhibitor and oxotremorine-M (OXO-M) as a muscarinic acetylcholine receptor (mAChRs) agonist, on the micturition reflex were evaluated by infusion cystometrography (CMG) in urethane-anaesthetized untreated rats or rats pretreated with capsaicin. RESULTS: Neostigmine injected i.c.v. increased bladder capacity (BC) and pressure threshold (PT) dose-dependently, with an increase in maximum voiding pressure (MVP) and a decrease in voiding efficiency (VE) at higher doses. Also, neostigmine injected i.t. increased the BC and PT dose-dependently without changing MVP or VE, and these effects were not apparent in capsaicin-pretreated rats. In both routes, atropine as an antagonist of mAChRs, but not mecamylamine as a nicotinic-AChR antagonist, almost completely antagonized the effects of neostigmine. The rank order of potencies of the antagonists for increasing effects of BC induced by 1 nmol of neostigmine was: pirenzepine (an M(1) mAChR antagonist) = atropine > 4-DAMP (an M(3) mAChR antagonist) " methoctramine (an M(2) mAChR antagonist) and tropicamide (an M(4) mAChR antagonist) via the i.c.v. route; and atropine > methoctramine > pirenzepine > tropicamide and 4-DAMP via the i.t. route, respectively. OXO-M injected via i.c.v. and i.t. had the same effects on BC, PT, MVP and VE as neostigmine by i.c.v. and i.t., respectively. CONCLUSIONS: These results indicate that activation of muscarinic cholinergic mechanisms by the cholinesterase inhibitor in the brain and spinal cord can inhibit the micturition reflex, mainly by affecting afferent pathways. These mAChR-induced inhibitory effects seem to be mediated through M(1)/M(3) receptor subtypes in the brain, while in the spinal cord, the M(1)/M(2) receptor subtypes might be involved in inhibitory effects, which are mediated via inhibition of mechanoceptive C-fibre afferent pathways.

Preconditioning stimuli that augment chromaffin cell secretion.

We have investigated here whether a preconditioned stimulation of nicotinic and muscarinic receptors augmented the catecholamine release responses elicited by supramaximal 3-s pulses of 100 muM acetylcholine (100ACh) or 100 mM K(+) (100K(+)) applied to fast-perifused bovine adrenal chromaffin cells. Threshold concentrations of nicotine (1-3 muM) that caused only a tiny secretion did, however, augment the responses elicited by 100ACh or 100K(+) by 2- to 3.5-fold. This effect was suppressed by mecamylamine and by Ca(2+) deprivation, was developed with a half-time (t(1/2)) of 1 min, and was reversible. The nicotine effect was mimicked by threshold concentrations of ACh, choline, epibatidine, and oxotremorine-M but not by methacholine. Threshold concentrations of K(+) caused lesser potentiation of secretion compared with that of threshold nicotine. The data are compatible with an hypothesis implying 1) that continuous low-frequency sympathetic discharge places chromaffin cells at the adrenal gland in a permanent "hypersensitive" state; and 2) this allows an explosive secretion of catecholamines by high-frequency sympathetic discharge during stress.

Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells.

The ability of G protein-coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH-SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1-100 microM), taurine influx was mediated exclusively by a Na(+)-dependent, high-affinity (K(m) = 2.5 microM) saturable transport mechanism (V(max) = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V(max), whereas the K(m) for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), also resulted in an attenuation of taurine influx (EC(50) approximately 0.7 microM). Although Oxo-M-mediated inhibition of taurine uptake could be observed under isotonic conditions (approximately 25-30%), the magnitude of inhibition was significantly enhanced by hypotonicity (approximately 55-60%), a result that also reflected a reduction in the V(max), but not the K(m), for taurine transport. Oxo-M-mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca(2+) but was independent of protein kinase C activity. In addition to Oxo-M, inclusion of either thrombin or sphingosine 1-phosphate also attenuated volume-dependent taurine uptake. The ability of Oxo-M to inhibit the influx of taurine was attenuated by 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid, an inhibitor of the volume-sensitive organic osmolyte and anion channel. 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid also prevented receptor-mediated changes in the efflux and influx of K(+) under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume-dependent efflux and uptake of taurine and that these events may be functionally coupled.

Activation of muscarinic cholinergic receptors on human SH-SY5Y neuroblastoma cells enhances both the influx and efflux of K+ under conditions of hypo-osmolarity.

The ability of receptor activation to regulate osmosensitive K+ fluxes (monitored as 86Rb+) in SH-SY5Y neuroblastoma has been examined. Incubation of SH-SY5Y cells in buffers rendered increasingly hypotonic by a reduction in NaCl concentration resulted in an enhanced basal efflux of Rb+ (threshold of release, 200 mOsM) but had no effect on Rb(+) influx. Addition of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), potently enhanced Rb+ efflux (EC50 = 0.45 microM) and increased the threshold of release to 280 mOsM. Oxo-M elicited a similarly potent, but osmolarity-independent, enhancement of Rb+ influx (EC50 = 1.35 microM). However, when incubated under hypotonic conditions in which osmolarity was varied by the addition of sucrose to a fixed concentration of NaCl, basal- and Oxo-M-stimulated Rb+ influx and efflux were demonstrated to be dependent upon osmolarity. Basal- and Oxo-M-stimulated Rb+ influx (but not Rb+ efflux) were inhibited by inclusion of ouabain or furosemide. Both Rb+ influx and efflux were inhibited by removal of intracellular Ca2+ and inhibition of protein kinase C activity. In addition to Oxo-M, agonists acting at other cell surface receptors previously implicated in organic osmolyte release enhanced both Rb+ efflux and influx under hypotonic conditions. Oxo-M had no effect on cellular K+ concentration in SH-SY5Y cells under physiologically relevant reductions in osmolarity (0-15%) unless K+ influx was blocked. Thus, although receptor activation enhances the osmosensitive efflux of K+, it also stimulates K+ influx, and the latter permits retention of K+ by the cells.

Coregulation of natively expressed pertussis toxin-sensitive muscarinic receptors with G-protein-activated potassium channels.

Many inhibitory neurotransmitters in the brain activate Kir3 channels by stimulating pertussis toxin (PTX)-sensitive G-protein-coupled receptors. Here, we investigated the regulation of native muscarinic receptors and Kir3 channels expressed in NGF-differentiated PC12 cells, which are similar to sympathetic neurons. Quantitative reverse transcription-PCR and immunocytochemistry revealed that NGF treatment significantly upregulated mRNA and protein for m2 muscarinic receptors, PTX-sensitive G alpha(o) G-proteins, and Kir3.2c channels. Surprisingly, these upregulated muscarinic receptor/Kir3 signaling complexes were functionally silent. Ectopic expression of m2 muscarinic receptors or Kir3.2c channels was unable to produce muscarinic receptor-activated Kir3 currents with oxotremorine. Remarkably, pretreatment with muscarinic (m2/m4) receptor antagonists resulted in robust oxotremorine-activated Kir3 currents. Thus, sustained cholinergic stimulation of natively expressed m2/m4 muscarinic receptors controlled cell surface expression and functional coupling of both receptors and Kir3 channels. This new pathway for controlling Kir3 signaling could help limit the potential harmful effects of excessive Kir3 activity in the brain.

Electrophysiological and immunofluorescence characterization of Ca(2+) channels of acutely isolated rat sphenopalatine ganglion neurons.

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.

Neuronally released acetylcholine acts on the M2 muscarinic receptor to oppose the relaxant effect of isoproterenol on cholinergic contractions in mouse urinary bladder.

We investigated whether M(2) muscarinic receptor activation opposes isoproterenol-induced relaxation in mouse urinary bladder and whether endogenous acetylcholine acts through a similar M(2) mechanism. When measured in urinary bladder from M(3) receptor knockout mice, the muscarinic agonist oxotremorine-M elicited only very weak contractions. In the presence of alpha,beta-methylene ATP (30 microM) and isoproterenol (1 microM), however, oxotremorine-M elicited a robust contractile response. This response was completely absent in bladder from M(2)/M(3) double knockout mice, indicating that activation of the M(2) receptor inhibits the relaxant effect of isoproterenol on the contraction to alpha,beta-methylene ATP. Similar results were obtained when prostaglandin F(2alpha) (5 microM) was used as the contractile agent but not when serotonin was used. Electrical field stimulation of the urinary bladder from wild-type mouse elicited contractions that were inhibited 20% by atropine and 40% by desensitization with alpha,beta-methylene ATP. When measured in the presence of alpha,beta-methylene ATP to desensitize the purinergic component of contraction, isoproterenol exhibited moderately greater relaxant activity in field-stimulated bladder from the M(2) knockout mouse compared with that observed in wild-type bladder. This differential relaxant effect of isoproterenol was greatly increased in the presence of physostigmine. In contrast, no differential effects were noted for isoproterenol in similar experiments on bladders from M(3) knockout and M(2)/M(3) double knockout mice in the presence of physostigmine. Our results suggest that neuronally released acetylcholine acts on the M(2) muscarinic receptor to inhibit the relaxant effect of isoproterenol on the minor, cholinergic component of contraction in the field-stimulated mouse urinary bladder.