Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Ionic selectivity of native ATP-activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia

1. The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP-evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole-cell patch clamp technique. 2. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na+ > Li+ > Cs+ > Rb+ > K+, and permeability ratios relative to Cs+ (P-X/P-Cs) ranging from 1. 11 to 0.86. 3. The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca2+ > Sr2+ > Ba2+ > Mn2+ > Mg2+. ATP-evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased. 4, The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size. 5. Acidification to pH 6.2 increased the ATP-induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti-P2X(2) and/or anti-P2X(4) but not anti-P2X(1) antibodies attenuated the ATP-evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X(2) and P2X(4) receptor subtypes expressed in rat submandibular neurones. 6. The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X2 receptors expressed in mammalian cells.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 �� 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 �� 2.4 and 6.7 �� 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 �� 0.97 and 7.5 �� 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

Triggering Solar-Powered Vehicle Activated Signs using Self Organising Maps with K-means

Solar-powered vehicle activated signs (VAS) are speed warning signs powered by batteries that are recharged by solar panels. These signs are more desirable than other active warning signs due to the low cost of installation and the minimal maintenance requirements. However, one problem that can affect a solar-powered VAS is the limited power capacity available to keep the sign operational. In order to be able to operate the sign more efficiently, it is proposed that the sign be appropriately triggered by taking into account the prevalent conditions. Triggering the sign depends on many factors such as the prevailing speed limit, road geometry, traffic behaviour, the weather and the number of hours of daylight. The main goal of this paper is therefore to develop an intelligent algorithm that would help optimize the trigger point to achieve the best compromise between speed reduction and power consumption. Data have been systematically collected whereby vehicle speed data were gathered whilst varying the value of the trigger speed threshold. A two stage algorithm is then utilized to extract the trigger speed value. Initially the algorithm employs a Self-Organising Map (SOM), to effectively visualize and explore the properties of the data that is then clustered in the second stage using K-means clustering method. Preliminary results achieved in the study indicate that using a SOM in conjunction with K-means method is found to perform well as opposed to direct clustering of the data by K-means alone. Using a SOM in the current case helped the algorithm determine the number of clusters in the data set, which is a frequent problem in data clustering.

RGS proteins reconstitute the rapid gating kinetics of G����-activated inwardly rectifying K+���channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of G��(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20���40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of ���regulators of G protein signaling��� (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor ��� GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent ���basal��� GIRK current was suppressed by ���40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of ���slow��� postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Effect of ��-amyloid block of the fast-inactivating K+ channel on intracellular Ca2+ and excitability in a modeled���neuron

��-Amyloid peptide (A��), one of the primary protein components of senile plaques found in Alzheimer disease, is believed to be toxic to neurons by a mechanism that may involve loss of intracellular calcium regulation. We have previously shown that A�� blocks the fast-inactivating potassium (A) current. In this work, we show, through the use of a mathematical model, that the A��-mediated block of the A current could result in increased intracellular calcium levels and increased membrane excitability, both of which have been observed in vitro upon acute exposure to A��. Simulation results are compared with experimental data from the literature; the simulations quantitatively capture the observed concentration dependence of the neuronal response and the level of increase in intracellular calcium.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles

<p>Purpose: Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina.</p><p>Methods: Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs).</p><p>Results: Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced Ca2+ influx in retinal VSMCs was reversed by the TRPV2 inhibitor tranilast and the nonselective TRPP1/V2 antagonist amiloride. Inhibitors of TRPC1, M7, V1, and V4 had no effect. Hypoosmotic stretch-activated cation currents were similar in Na+ and Cs+ containing solutions suggesting no contribution by TRPP1 channels. Direct plasma membrane stretch triggered cation current activity that was blocked by tranilast and specific TRPV2 pore-blocking antibodies and mimicked by the TRPV2 activator, ��9-tetrahydrocannabinol. Preincubation of retinal arterioles with TRPV2 blocking antibodies prevented the development of myogenic tone.</p><p>Conclusions: Our results suggest that retinal VSMCs express a range of mechanosensitive TRP channels, but only TRPV2 appears to contribute to myogenic signaling in this vascular bed.</p>

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic ��-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the ��1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

Redox properties of the adenoside triphosphate-sensitive K+ channel in brain mitochondria

Brain mitochondrial ATP-sensitive K+ channel (mito-K-ATP) opening by diazoxide protects against ischemic damage and excitotoxic cell death. Here we studied the redox properties of brain mito-K-ATP. Mito-K-ATP activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mito-K-ATP activation in isolated brain mitochondria significantly prevented H2O2 release by these organelles but did not change Ca2+ accumulation capacity. Interestingly, the activity of mito-K-ATP was highly dependent on redox state. The thiol reductant mercaptopropionylglycine prevented mito-K-ATP activity, whereas exogenous ROS activated the channel. In addition, the use of mitochondrial substrates that led to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K+ transport activity through mito-K-ATP. Altogether, our results indicate that brain mito-K-ATP is a redox-sensitive channel that controls mitochondrial ROS release. (c) 2008 Wiley-Liss, Inc.

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg2+-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable K-d, of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 mu M free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.

Granulocyte-Colony Stimulating Factor (G-CSF) induces mechanical hyperalgesia via spinal activation of MAP kinases and PI(3)K in mice

Granulocyte-colony stimulating factor (G-CSF) is a current pharmacological approach to increase peripheral neutrophil counts after anti-tumor therapies. Pain is most relevant side effect of G-CSF in healthy volunteers and cancer patients. Therefore, the mechanisms of G-CSF-induced hyperalgesia were investigated focusing on the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase). JNK (Jun N-terminal Kinase) and p38, and PI(3)K (phosphatidylinositol 3-kinase). G-CSF induced dose (30-300 ng/paw)-dependent mechanical hyperalgesia, which was inhibited by local post-treatment with morphine. This effect of morphine was reversed by naloxone (opioid receptor antagonist). Furthermore, G-CSF-induced hyperalgesia was inhibited in a dose-dependent manner by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmanin) inhibitors. The co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited G-CSF-induced hyperalgesia. Concluding, in addition to systemic opioids, peripheral opioids as well as spinal treatment with MAP kinases and PI(3)K inhibitors also reduce G-CSF-induced pain. (C) 2011 Elsevier Inc. All rights reserved.

Twisted sl(3, C)(k)((2)) current algebra: free field representation and screening currents

Motivated by application of twisted current algebra in description of the entropy of Ads(3) black hole, we investigate the simplest twisted current algebra sl(3, c)(k)((2)). Free field representation of the twisted algebra, and the corresponding twisted Sugawara energy-momentum tensor are obtained by using three (beta, gamma) pairs and two scalar fields. Primary fields and two screening currents of the first kind are presented. (C) 2001 Published by Elsevier Science B.V.

An ATP-sensitive K+ conductance in dissociated neurones from adult rat intracardiac ganglia

1. An ATP-sensitive K+ (K-ATP) conductance has been identified using the perforated patch recording configuration in a population (52%) of dissociated neurones from adult rat intracardiac ganglia. The presence of the sulphonylurea receptor in approximately half of the intracardiac neurones was confirmed by labelling with fluorescent glibenclamide-BODIPY. 2. Under current clamp conditions in physiological solutions, leveromakalim (10 muM) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide. 3. Under voltage clamp conditions in symmetrical (140 mM) K+ solutions, hath application of levcromakalim evoked an inward current with a density of similar to8 pA pF(-1) at -50 mV and a slope conductance of similar to9 nS, which reversed close to the potassium equilibrium potential (E-K). Cell dialysis with an ATP-free intracellular solution also evoked an inward current, which was inhibited by tolbutamide. 4. Bath application of either glibenclamide (10 muM) or tolbutamide (100 muM) depolarized adult intracardiac neurones by 3-5 mV, suggesting that a K-ATP conductance is activated under resting conditions and contributes to the resting membrane potential. 5. Activation of a membrane current by levcromakalim leas concentration dependent, with an EC50 of 1.6 muM. Inhibition of the levcromakalim-activated current by glibenclamide leas also concentration dependent, with an IC50 of 55 nM. 6. Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (P-O2 similar to 16 mmHg) also activated a membrane current. These currents exhibited similar I-P characteristics to the levcroinakalim-induced current and were inhibited by glibenclamide. 7. Activation of K-ATP channels in mammalian intracardiac neurones may contribute to changes in neural regulation of the mature heart and. cardiac function during ischaemia-reperfusion.