Neuroscience. What genes tell us about nicotine addiction.
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Animals↗
Biomedical subjects
R C Hogg
Publications and source records attributed to R C Hogg.
Nicotinic acetylcholine receptors as drug targets.
While it has long been documented that nicotine contained in tobacco leaves gives rise to major public health problems it has also been observed that this alkaloid can have beneficial effects. However, it is only with the identification of a family of genes coding for the neuronal nicotinic acetylcholine receptors and increased knowledge of their expression and function in the central nervous system that these receptors have received attention concerning their potential as drug targets. In light of the latest findings about nicotinic acetylcholine receptors and their involvement in disease states we review the possibility to design new drugs targeted to these ligand-gated channels. Beneficial and possible undesirable actions of agonists, antagonists and allosteric modulators are discussed and placed in perspective of our most recent knowledge.
Alzheimer Disease↗
Nicotinic acetylcholine receptors: from structure to brain function.
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and can be divided into two groups: muscle receptors, which are found at the skeletal neuromuscular junction where they mediate neuromuscular transmission, and neuronal receptors, which are found throughout the peripheral and central nervous system where they are involved in fast synaptic transmission. nAChRs are pentameric structures that are made up of combinations of individual subunits. Twelve neuronal nAChR subunits have been described, alpha2-alpha10 and beta2-beta4; these are differentially expressed throughout the nervous system and combine to form nAChRs with a wide range of physiological and pharmacological profiles. The nAChR has been proposed as a model of an allosteric protein in which effects arising from the binding of a ligand to a site on the protein can lead to changes in another part of the molecule. A great deal is known about the structure of the pentameric receptor. The extracellular domain contains binding sites for numerous ligands, which alter receptor behavior through allosteric mechanisms. Functional studies have revealed that nAChRs contribute to the control of resting membrane potential, modulation of synaptic transmission and mediation of fast excitatory transmission. To date, ten genes have been identified in the human genome coding for the nAChRs. nAChRs have been demonstrated to be involved in cognitive processes such as learning and memory and control of movement in normal subjects. Recent data from knockout animals has extended the understanding of nAChR function. Dysfunction of nAChR has been linked to a number of human diseases such as schizophrenia, Alzheimer's and Parkinson's diseases. nAChRs also play a significant role in nicotine addiction, which is a major public health concern. A genetically transmissible epilepsy, ADNFLE, has been associated with specific mutations in the gene coding for the alpha4 or beta2 subunits, which leads to altered receptor properties.
Animals↗
Regulating the regulators: the role of nicotinic acetylcholine receptors in human epilepsy.
Recent advances in genetic mapping have linked several epilepsies to mutations in genes coding for ion channel proteins. Mutations in genes coding for the alpha(4) and beta(2) subunits of the nicotinic acetylcholine receptor have been found in some families suffering from autosomal dominant nocturnal frontal lobe epilepsy. Expression of mutant alleles in the Xenopus oocyte expression system has allowed functional characterization of these mutant receptors at the cellular level. The challenge is now to design therapeutic strategies based on this knowledge of how receptor function is altered in certain epilepsies.
Animals↗
Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons.
The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, Ih, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, IK(IR), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+ Using the perforated-patch recording configuration, an ATP-sensitive K+ (KATP) conductance was identified in > or = 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion.
Adenosine Triphosphate↗
Ciguatoxin-induced oscillations in membrane potential and action potential firing in rat parasympathetic neurons.
The actions of ciguatoxins from the Pacific (P-CTX-1) and Caribbean (C-CTX-1) regions were investigated in isolated parasympathetic neurons from rat intracardiac ganglia using patch-clamp recording techniques. Under current-clamp conditions, bath application of P-CTX-1 (1-10 nm) or C-CTX-1 (10-30 nm) caused a gradual depolarization that was accompanied by oscillation of the membrane potential leading to tonic action potential firing. Membrane potential oscillations were observed between -45 and -60 mV and had an amplitude of 10-20 mV and a mean frequency of 10 Hz. Oscillation frequency was temperature-dependent with a Q10 of 2.0. Membrane oscillations were temporarily inhibited by hyperpolarizing current pulses and potentiated by weak depolarizing current pulses. The amplitude of oscillations was reduced upon lowering the external Na+ concentration and inhibited by tetrodotoxin (TTX), tetracaine or Zn2+. Tetraethylammonium, 4-aminopyridine, Cs+, Cd2+, Ba2+, 1,4,4'-diothiocyanato-2,2'-stilbenedisulphonic acid (DIDS) and ouabain had no effect on the CTX-1-induced membrane depolarization and oscillations. Brevetoxin (PbTx-3, 100 nm), in contrast to CTX-1, caused a membrane depolarization that was not associated with oscillation of the membrane potential. Under voltage-clamp conditions, P-CTX-1 inhibited the peak amplitude of the voltage-dependent Na+ current and shifted the activation curve to more negative potentials, but membrane oscillations were not seen in this configuration. These results suggest that ciguatoxins cause oscillation of the membrane potential in mammalian autonomic neurons by modifying the activation and inactivation properties of a population of TTX-sensitive Na+ channels.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
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, levcromakalim (10 microM) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide. 3. Under voltage clamp conditions in symmetrical (140 mM) K(+) solutions, bath application of levcromakalim evoked an inward current with a density of 8 pA pF(-1) at -50 mV and a slope conductance of approximately 9 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 microM) or tolbutamide (100 microM) 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 was concentration dependent with an EC(50) of 1.6 microM. Inhibition of the levcromakalim-activated current by glibenclamide was also concentration dependent with an IC(50) of 55 nM. 6. Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (P(O2) approximately 16 mmHg) also activated a membrane current. These currents exhibited similar I-V characteristics to the levcromakalim-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.
Adenosine Triphosphate↗
Developmental changes in hyperpolarization-activated currents I(h) and I(K(IR)) in isolated rat intracardiac neurons.
The hyperpolarization-activated nonselective cation current, I(h), was investigated in neonatal and adult rat intracardiac neurons. I(h) was observed in all neurons studied and displayed slow time-dependent rectification. I(h) was isolated by blockade with external Cs(+) (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I(h) was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I(h) was sensitive to changes in external Na(+) and K(+) concentrations. An inwardly rectifying K(+) current, I(K(IR)), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba(2+). I(K(IR)) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I(K(IR)) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K(+) rectifier described in other mammalian autonomic neurons. I(K(IR)) was sensitive to changes in external K(+), whereby raising the external K(+) concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na(+) had no effect on the reversal potential or amplitude of I(K(IR)). I(K(IR)) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I(h) may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.
Age Factors↗
Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor.
The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication. In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC(50) values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC(50) value of 375 nM and a maximally effective concentration caused 91% block. [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in approximately 80% of neurons, with an IC(50) value of 1.4 nM and 46% maximal block of the total current. The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the alpha-conotoxins PnIA or PnIB, and by mecamylamine. (1)H NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and 11 of PnIA and PnIB influence potency and determine selectivity among alpha7 and other nAChR subtypes, including alpha3beta2 and alpha3beta4. Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.
Amino Acid Sequence↗
Mechanisms of verapamil inhibition of action potential firing in rat intracardiac ganglion neurons.
The effects of verapamil and related phenylalkylamines on neuronal excitability were investigated in isolated neurons of rat intracardiac ganglia using whole-cell perforated patch-clamp recording. Verapamil (>/=10 microM) inhibits tonic firing observed in response to depolarizing current pulses at 22 degrees C. The inhibition of discharge activity is not due to block of voltage-dependent Ca2+ channels because firing is not affected by 100 microM Cd2+. The K+ channel inhibitors charybdotoxin (100 nM), 4-aminopyridine (0.5 mM), apamin (30-100 nM), and tetraethylammonium ions (1 mM) also have no effect on firing behavior at 22 degrees C. Verapamil does not antagonize the acetylcholine-induced inhibition of the muscarine-sensitive K+ current (M-current) in rat intracardiac neurons. Verapamil inhibits the delayed outwardly rectifying K+ current with an IC50 value of 11 microM, which is approximately 7-fold more potent than its inhibition of high voltage-activated Ca2+ channel currents. These data suggest that verapamil inhibits tonic firing in rat intracardiac neurons primarily via inhibition of delayed outwardly rectifying K+ current. Verapamil inhibition of action potential firing in intracardiac neurons may contribute, in part, to verapamil-induced tachycardia.
4-Aminopyridine↗
Ciguatoxin (CTX-1) modulates single tetrodotoxin-sensitive sodium channels in rat parasympathetic neurones.
The actions of the marine neurotoxin, ciguatoxin-1 (CTX-1), were investigated in isolated parasympathetic neurones from neonatal rat intracardiac ganglia using patch-clamp recording techniques. Under current clamp conditions, bath application of 1-10 nM CTX-1 caused gradual membrane depolarization and tonic action potential firing. Action potential firing ceased with depolarization beyond approximately -35 mV and application of 300 nM tetrodotoxin (TTX) repolarized the cell to its control resting potential. In cell-attached membrane patches, 1-10 nM CTX-1 in the patch pipette markedly increased the open probability of single TTX-sensitive Na+ channels in response to depolarizing voltage steps but did not alter the unitary conductance (10 pS) or reversal potential. Under steady-state conditions, CTX-1 caused spontaneous opening of single Na+ channels which did not inactivate at hyperpolarized membrane potentials. CTX-1 increases neuronal excitability by shifting the voltage of activation of TTX-sensitive Na+ channels to more negative potentials.
Action Potentials↗
Effect of frusemide, ethacrynic acid and indanyloxyacetic acid on spontaneous Ca-activated currents in rabbit portal vein smooth muscle cells.
1. The effect of frusemide, ethacrynic acid and indanyloxyacetic acid was investigated on spontaneous calcium-activated chloride (ICl(Ca)) and potassium currents (IK(Ca)) in rabbit portal vein cells with the perforated patch technique. 2. Frusemide (0.3-1.0 x 10(-3) M) reduced the amplitude of spontaneous transient inward chloride currents (STICs) in a concentration-dependent manner. The degree of inhibition on STIC amplitude was similar between -50 and +30 mV and frusemide did not alter the STIC reversal potential (Erev). 3. The voltage-dependent exponential decay of STICs, which is thought to represent closure of chloride channels, was not altered by frusemide. 4. The amplitude and frequency of spontaneous potassium outward currents (STOCs) were not altered by frusemide. Since both STICs and STOCs are activated by calcium released from intracellular stores these data indicate that frusemide may block directly ICl(Ca). 5. Ethacrynic acid (2-5 x 10(-4) M) decreased the amplitude of STICs in a concentration-dependent manner by a similar amount at potentials of -50 to +30 mV but did not alter the STIC Erev. However, these concentrations of ethacrynic acid also reduced STOC amplitude and 5 x 10(-4) M ethacrynic acid evoked a sustained outward current in most cells at 0 mV; thus ethacrynic acid has a more complex action than simple block of ICl(Ca). 6. Indanyloxyacetic acid reduced both STIC amplitude and decay time without affecting STOCs and thus also seems to inhibit directly ICl(Ca). It is discussed whether block of ICl(Ca) mediates the vasodilator effect of these agents.
Animals↗
Synergistic action of histamine and adenosine triphosphate on the response to noradrenaline in rabbit pulmonary artery smooth muscle cells.
The interaction between histamine, adenosine triphosphate (ATP) and noradrenaline was studied with the perforated-patch technique in single cells isolated from the rabbit pulmonary artery. In these cells all of the agents activated caffeine-sensitive currents. In potassium-free conditions at a holding potential of -50 mV bath applied histamine, in concentrations that did not produce a response, greatly enhanced the magnitude of inward currents evoked by ionophoretic application of noradrenaline. These inward currents were calcium-activated chloride currents, ICl(Ca). In potassium-containing solutions at a holding potential of 0 mV, bath applied histamine potentiated the outward currents (calcium-activated potassium currents (IK(Ca)) induced by noradrenaline. This synergistic action was rapid in onset, sustained during the continued presence of histamine and reversible. Bath application of noradrenaline inhibited the response to ionophoretically applied noradrenaline but not the caffeine-induced currents. ATP also stimulated ICl(Ca) and IK(Ca) through a mechanism dependent on the caffeine-sensitive intracellular calcium store and also potentiated the currents activated by noradrenaline. It is concluded that one explanation for the phenomenon of potentiation in smooth muscle is convergence of several distinct pharmacological receptors to a common cellular mechanism.
Adenosine Triphosphate↗
Action of niflumic acid on evoked and spontaneous calcium-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.
1. The action of niflumic acid was studied on spontaneous and evoked calcium-activated chloride (ICl(Ca)) and potassium (IK(Ca)) currents in rabbit isolated portal vein cells. 2. With the nystatin perforated patch technique in potassium-containing solutions at a holding potential of -77 mV (the potassium equilibrium potential), niflumic acid produced a concentration-dependent inhibition of spontaneous transient inward current (STIC, calcium-activated chloride current) amplitude. The concentration to reduce the STIC amplitude by 50% (IC50) was 3.6 x 10(-6) M. 3. At -77 mV holding potential, niflumic acid converted the STIC decay from a single exponential to 2 exponential components. In niflumic acid the fast component of decay was faster, and the slow component was slower than the control decay time constant. Increasing the concentration of niflumic acid enhanced the decay rate of the fast component and reduced the decay rate of the slow component. 4. The effect of niflumic acid on STIC amplitude was voltage-dependent and at -50 and +50 mV the IC50 values were 2.3 x 10(-6) M and 1.1 x 10(-6) M respectively (cf. 3.6 x 10(-6) M at -77 mV). 5. In K-free solutions at potentials of -50 mV and +50 mV, niflumic acid did not induce a dual exponential STIC decay but just increased the decay time constant at both potentials in a concentration-dependent manner. 6. Niflumic acid, in concentrations up to 5 x 10(-5) M, had no effect on spontaneous calcium-activated potassium currents. 7. Niflumic acid inhibited noradrenaline- and caffeine-evoked IO(Ca) with an ICM50 of 6.6 x 10-6 M, i.e.was less potent against evoked currents compared to spontaneous currents. In contrast niflumic acid(2 x 10-6 M-5 x 105 M) increased noradrenaline- and caffeine-induced IK(ca).8. The results are discussed with respect to the mechanism of block of ICl(Ca) by niflumic acid and its suitability as a pharmacological tool for assessing the role of Ic(ca) in physiological mechanisms.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Effects of Cl channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.
1. The effects of some chloride channel antagonists were studied on the calcium-activated chloride current (ICl(Ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique. 2. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium-activated chloride currents) in a concentration-dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 x 10(-4) M and 6.4 x 10(-4) M for DIDS and SITS, respectively. This effect was not voltage-dependent. 3. The time constant of decay of STICs (tau), which is voltage-dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on tau was similar at holding potentials of -50 and +50 mV. 4. These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium-activated potassium currents). 5. DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 x 10(-4) M and 1.8 x 10(-3) M, respectively. 6. DIDS and SITS increased the calcium-activated potassium current (IK(Ca) evoked by noradrenaline and caffeine by 3-6 fold. 7. Anthracene-9-carboxylic acid (A-9-C) inhibited STICs in a voltage-dependent fashion and was about 3 fold more active at +50 mV than at -50 mV. A-9-C increased STIC tau and this effect was enhanced by depolarization. 8. A-9-C also inhibited caffeine-evoked IC1(ca) but less potently than STICs and also increased the evoked IK(ca) without altering spontaneous IK(Ca).9. The results from the present work are compared with the pharmacology of other chloride conductances and the mechanism of action of the chloride channel antagonists in vascular smooth muscle is discussed.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Properties of spontaneous inward currents in rabbit pulmonary artery smooth muscle cells.
Spontaneous inward and outward currents were studied with perforated patch recording in freshly dispersed rabbit pulmonary artery smooth muscle cells. With physiological potassium concentrations, spontaneous outward and inward currents were recorded at negative membrane potentials. Ion substitution experiments revealed that the outward and inward currents were respectively potassium and chloride conductance increases. Both conductances were abolished by bath application of caffeine (2-10 mM), which releases calcium from internal stores. The rise time and half-decay time of spontaneous potassium currents were both about 25 ms. The spontaneous chloride current has a rise time of 30 ms and decayed exponentially with a time constant (tau) of 70 ms at -50 mV. The tau value was increased by depolarization and increased e-fold for a change of 99 mV in membrane potential. In every cell examined when the spontaneous currents occurred as biphasic events, typically between -20 mV and -40 mV, outward currents preceded inward currents in over 90% of these events whereas the inward current always preceded the outward current in caffeine- and noradrenaline-evoked responses. An explanation for these data is that there may be localization of some chloride channels with respect to the caffeine-sensitive calcium store.
Animals↗
A monovalent ion-selective cation current activated by noradrenaline in smooth muscle cells of rabbit ear artery.
Membrane currents were recorded with the perforated-patch method with a low-chloride (35 mM) pipette solution in isolated smooth muscle cells of the rabbit ear artery. At a holding potential of -50 mV in potassium-free conditions spontaneous inward single-channel currents were observed and noradrenaline evoked a noisy inward current, which appeared to be comprised of the spontaneous currents. The reversal potential (Vr) of the spontaneous channel and noradrenaline-induced current was not affected in anion-substitution experiments but Vr was altered when external Na+ was replaced with choline or TRIS. The relationship between clamp potential and spontaneous single-channel current amplitude was linear and the mean unitary conductance was 28 pS. Caffeine, which releases calcium from the sarcoplasmic reticulum, and the calcium ionophore ionomycin activated the cation current and also blocked the response to noradrenaline. Spontaneous channel current activity and the noradrenaline-induced current were blocked when external NaCl was replaced with 89 mM CaCl2. The response to noradrenaline was blocked by prazosin but was not affected by yohimbine and therefore the response is mediated by alpha 1-adrenoceptors. It is concluded that in rabbit ear artery smooth muscle cells there is a calcium-activated cation channel of 28 pS conductance, which is relatively impermeable to calcium but can be activated by noradrenaline.
Animals↗
Time course of spontaneous calcium-activated chloride currents in smooth muscle cells from the rabbit portal vein.
1. The time course of spontaneous calcium-activated chloride currents was studied with the perforated patch technique in freshly dispersed smooth muscle cells from the rabbit portal vein. 2. In potassium-containing solutions the spontaneous transient outward current (STOC, a calcium-activated potassium current) was more commonly recorded than spontaneous transient inward currents (STICs, a calcium-activated chloride current). In addition the duration of STOCs was much briefer (about 100 ms) than the duration of STICs (about 400 ms). 3. The decay of STICs could be described by a single exponential but the STOC decay appeared to be more complex. The decay time constant of STICs was not determined significantly by amplitude. 4. The time constant of decay of STICs (tau) was 86 ms at -50 mV and was increased by depolarization. Between -90 and +50 mV the relationship between tau and membrane potential was exponential and tau changed e-fold for a change of membrane potential of 120 mV. 5. The I-V relationship of STIC amplitude was linear between -10 and +50 mV but at more negative potentials the chord conductance was reduced and the I-V relationship exhibited negative slope conductance between -50 and -90 mV. 6. There was good agreement between the STIC tau values and the exponential relaxations to voltage steps evoked during caffeine-induced calcium-activated chloride currents. 7. In the presence of the chloride channel blocking agent anthracene-9-carboxylic acid the STIC amplitude was reduced and tau was increased. This effect was voltage dependent with a much greater effect at positive potentials. 8. The evidence suggests that the decay of STICs represents closure of chloride channels and tau approximates to the channel mean open time.
Animals↗