Search

Cl−channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl−conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl−current in freshly isolated guinea pig DSM cells. The Cl−current demonstrated outward rectification arising from voltage-dependent gating of Cl−channels rather than the Cl−transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl−current providing strong evidence that volume-regulated anion channels do not contribute to the Cl−current in DSM cells. The Cl−current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] analog [PI(4,5)P2-diC8] increased the average Cl−current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl−current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P2-diC8, an intriguing finding that underscores the complex nature of Cl−channel expression and function in DSM cells. Removal of both extracellular Ca2+and Mg2+did not affect the DSM whole cell Cl−current, whereas Gd3+(1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl−channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd3+, and PI(4,5)P2.

Transient receptor potential melastatin 4 (TRPM4) channels are Ca2+-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder.

Members of the transient receptor potential (TRP) channel superfamily, including the Ca2+-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca2+ imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca2+ imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca2+ levels. 9-Phenanthrol (0.1–30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1–7 μM and 70–80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5–50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.

alpha4beta2 neuronal nicotinic receptors (nAChRs) can exist in high and low sensitivity states possibly due to distinct stoichiometries during subunit assembly: (alpha4)(2)(beta2)(3) pentamer (high sensitivity, HS) and (alpha4)(3)(beta2)(2) pentamer (low sensitivity, LS). To determine if there is a linkage between HS or LS states and receptor-mediated responses in brain, we profiled several clinically studied alpha4beta2* nAChR agonists for the displacement of radioligand binding to alpha4beta2 [(3)H]-cytisine sites in rat brain membranes, effects on stimulation of [(3)H]-dopamine release from slices of rat prefrontal cortex and striatum, and activation of HS and LS human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Binding affinities (pK(i)) and potency (pEC(50)) values for [(3)H]-dopamine release closely correlated with a rank order: varenicline(-)-nicotineAZD3480dianicline congruent with ABT-089. Further, a good correlation was observed between [(3)H]-dopamine release and HS alpha4beta2 pEC(50) values, but not between [(3)H]-dopamine release and LS alpha4beta2. The relative efficacies of the agonists ranged from full to partial agonists. Varenicline behaved as a partial agonist in stimulating [(3)H]-dopamine release and activating both HS and LS alpha4beta2 nAChRs expressed in oocytes. Conversely, ABT-089, AZD3480 and dianicline exhibited little efficacy at LS alpha4beta2 (5%), were more effective at HS alpha4beta2 nAChRs, and in stimulating cortical and striatal [(3)H]-dopamine releaseor=30%. In the presence of alpha-conotoxin MII to block alpha6beta2* nAChRs, the alpha4beta2* alpha-conotoxin-insensitive [(3)H]-dopamine release stimulated by these ligands correlates well with their interactions at HS, but not LS. In summary, this study provides support for HS alpha4beta2* nAChR involvement in neurotransmitter release.

Background and purpose: The α7 nicotinic acetylcholine receptor (nAChR) has attracted considerable interest as a target for cognitive enhancement in schizophrenia and Alzheimer's Disease. However, most recently described α7 agonists are derived from the quinuclidine structural class. Alternatively, the present study identifies tilorone as a novel α7-selective agonist and characterizes analogues developed from this lead. Experimental approach: Activity and selectivity were determined from rat brain α7 and α4β2 nAChR binding, recombinant nAChR activation, and native α7 nAChR mediated stimulation of ERK1/2 phosphorylation in PC12 cells. Key results: Tilorone bound α7 nAChR (IC50 110 nM) with high selectivity relative to α4β2 (IC50 70 000 nM), activated human α7 nAChR with an EC50 value of 2.5 μM and maximal response of 67% relative to acetylcholine, and showed little agonist effect at human α3β4 or α4β2 nAChRs. However, the rat α7 nAChR maximal response was only 34%. Lead optimization led to 2-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-xanthen-9-one (A-844606) with improved binding (α7 IC50 11 nM, α4β2 IC50>30 000 nM) and activity at both human and rat α7 nAChR (EC50s 1.4 and 2.2 μM and apparent efficacies 61 and 63%, respectively). These compounds also activated native α7 nAChR, stimulating ERK1/2 phosphorylation in PC12 cells. Conclusions and implications: Tilorone, known as an interferon inducer, is a selective α7 nAChR agonist, suggesting utility of the fluorenone pharmacophore for the development of α7 nAChR selective agonists. Whether α7 stimulation mediates interferon induction, or whether interferon induction may influence the potential anti-inflammatory properties of α7 nAChR agonists remains to be elucidated. British Journal of Pharmacology (2008) 153, 1054–1061; doi:10.1038/sj.bjp.0707649; published online 24 December 2007

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

The α7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel α7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki= 10.8 nm) and human (Ki= 16.7 nm) α7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the α7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that α7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of α7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked α7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that α7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

Interstitial cells of Cajal (ICC) form networks that intercalate between the enteric nervous system and smooth muscle cells and play a fundamental role in the control of gastrointestinal motility by initiating rhythmic electrical activity. In this report, we used a method to examine the physiological and morphological properties of ICC in living, intact tissues. ACK2, an anti-Kit antibody, was conjugated to a fluorescent probe and used to identify individual ICC for intracellular electrical recordings, to record changes in intracellular calcium concentration using fluorescent dyes and for confocal microscopy. Cyclic changes in intracellular calcium concentration were recorded in ICC with a frequency similar to the electrical slow wave. In addition, injection of a fluorescent dye into single ICC enabled the three-dimensional reconstruction of single myenteric plexus ICC within the intact network. The data show that ICC in intact networks from the myenteric plexus region in living tissues in the guinea-pig antrum exhibit an electrical slow wave, and that intracellular calcium oscillates at a frequency similar to the slow wave.

We investigated whether muscarinic receptor (MR) activation increases human urinary bladder smooth muscle (UBSM) excitability by directly or indirectly inhibiting the large conductance Ca2+-activat...

Interstitial cells of Cajal (ICCs) form a network of cells between the external longitudinal and circular muscle layers at the level of the Auerbach's plexus in the mammalian small intestine. These cells express the Kit receptor tyrosine kinase and are essential for intestinal pacemaker activity. W mutant mice carrying structural mutations in the Kit gene lack both the network of ICCs and intestinal pacemaker activity. We were interested in the developmental origin of the cells that make up the network of ICCs. In addition, the specific stages of ICC development that require a functional Kit receptor have not been characterized. We show that ICCs originate from mesenchymal progenitor cells that coexpress both Kit and smooth muscle myosin heavy chain, a marker specific for smooth muscle, during embryogenesis. ICC and longitudinal smooth muscle lineages begin to diverge late in gestation. Embryos homozygous for the regulatory Wbanded (Wbd) mutation do not express Kit in these mesenchymal progenitor cells. Nevertheless, Wbd/Wbd mice display a normal network of ICCs and normal smooth muscle layers at postnatal day 5 (p5). Adult Wbd/Wbd mice lack a functional ICC network and intestinal pacemaker activity due to a failure of the ICCs to increase in numbers after p5. These data suggest a common developmental origin of the ICCs and the longitudinal smooth muscle layers in the mammalian small intestine and show that Kit expression is necessary for the postnatal development and proliferation of ICCs but not for the initial cell lineage decision toward an ICC fate during embryogenesis or for smooth muscle development.

Three preparations of humic material (a commercial humic acid and material isolated from soil and from water) were analysed by electrophoresis on polyacrylamide gradient gel slabs. All gave similar patterns showing four bands of material of molecular weights apparently ranging from a few hundred to about 20,000 as estimated by comparing their mobilities with those of protein markers. The high molecular weight material from bleached kraft mill effluent (BKME) showed similar patterns with the addition of completely unresolved material of molecular weight up to about 100,000. Electrophoresis on polyacrylamide slabs may prove valuable for the study of humic substances and other ill-defined polymeric materials.

The pacemaker activity in the mammalian gut is responsible for generating anally propagating phasic contractions. The cellular basis for this intrinsic activity is unknown. The smooth muscle cells of the external muscle layers and the innervated cellular network of interstitial cells of Cajal, which is closely associated with the external muscle layers of the mammalian gut, have both been proposed to stimulate pacemaker activity. The interstitial cells of Cajal were identified in the last century but their developmental origin and function have remained unclear. Here we show that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells of Cajal associated with Auerbach's nerve plexus and intestinal pacemaker activity.