Channel Ligands Research Articles

The relationship between the NMDA receptor NR1 subunit mRNA and [3H]MK-801 binding in the embryonic and early postnatal rat CNS

The relationship between the NMDA receptor NR1 subunit mRNA and the binding of the NMDA channel ligand [3H]MK-801 was studied during the late embryonic and early postnatal period in various regions of the rat CNS. NR1 mRNA was detectable at embryonic day 18 in all regions analysed and specific binding sites for [3H]MK-801 were present in the cerebral cortex at least from the day of birth, with an unaltered affinity up to postnatal day 15. In the cortex, hippocampus and hypothalamus there was a pronounced increase in binding between postnatal day 8 and 15, while the NR1 mRNA levels during this period were unchanged or even decreased. A more parallel developmental profile of mRNA content and binding levels were seen in the medulla and spinal cord. In the cerebellum the profile was different compared to the other regions, with a clear increase in NR1 mRNA between postnatal day 8 and 15 without any change in the [3H]MK-801 binding level. We conclude that even though the NR1 subunit alone possesses all the properties characteristic for the NMDA receptor, we cannot find a clear relationship between the level of its mRNA and the amount of [3H]MK-801 binding in the developing rat CNS.

The influx of Ca 2+ and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca 2+ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca 2+] i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca 2+ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca 2+] i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca 2+ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca 2+] i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca 2+ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.

Modulation of vascular K ATP channels in hypothyroidism

The role of vascular K ATP channels in hypothyrodism-induced decrease in myogenic activity of rat portal vein was examined by using pharmacologically relevant concentrations of K + channel ligands. As compared to controls, a significant decrease in the myogenic tone and noradrenaline (10 −9–10 −5 M)-induced contractions was observed in portal veins from hypothyroid rats. In both euthyroid and hypothyroid states, pinacidil (10 −9–10 −5 M) and cromakalim ((10 −9–10 −5 M) caused concentration-related inhibition of the myogenic tone (frequency and amplitude). However, hypothyroidism caused a leftward shift in the concentration-response curves of the K + channel openers with a corresponding decrease in their IC 50 values both in the absence and presence of the K ATP channel blocker, glibenclamide (10 −7 M). Further, concentration-dependent increase in the frequency of myogenic tone by glibenclamide (10 −8–3 × 10 −6 M) was greater in tissues from hypothyroid rats (EC 50 = 2.07 × 10 −7 M; 95% CL, 1.06−4.05 × 10 −7 M) in comparison to controls (EC 50 = 8.07 × 10 −7 M; 95% CL, 0.53−1.22 × 10 −6 M). These results suggest that a decrease in the myogenic tone of rat portal vein may possibly be related to an enhanced opening of the K ATP channels in hypothyroidism.

Modulation of nicotine-evoked [3H]dopamine release from rat striatal synaptosomes by voltage-sensitive calcium channel ligands

The calcium channel subtypes mediating nicotine-evoked [3H]dopamine release from rat striatal synaptosomes were probed with L-, N-, and P-type calcium channel ligands. Responses to nicotine were blocked by the peptides omega-conotoxin GVIA and omega-agatoxin IVA. The affinity constants for these compounds were consistent with their actions at N- and P-type channels, respectively. Together, these channels mediate at least 90% of the calcium-dependent response to nicotine. The L-type antagonists nifedipine, verapamil, and nicardipine were also effective blockers of nicotine-evoked release with maximal effects of 80-100% inhibition. However, these effects occurred at concentrations 2-3 orders of magnitude higher than those necessary to block L-type channels. Moreover, Bay K8644, an L-type agonist, also blocked nicotine-evoked release. Together, these findings argue strongly against the involvement of L-type channels.

A-85380 [3-(2( S)-azetidinylmethoxy) pyridine]: In Vitro pharmacological properties of a novel, high affinity α4β2 nicotinic acetylcholine receptor ligand

The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2( S)-azetidinylmethoxy) pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human α4β2 nAChR subtype ( K i = 0.05 ± 0.01 nM) relative to the human α7 ( K i = 148 ± 13 nM) and the muscle α1β1dg subtype expressed in Torpedo electroplax ( K i = 314 ± 12 nM). The R-enantiomer of A-85380, A-159470, displays little enantioselectivity towards the α4β2 and α1β1δγ subtypes but does display 12-fold enantioselectivity towards the α7 subtype ( K i = 1275 ± 199 nM). (+)- and (−)-Epibatidine display similar potencies at the human human α4β2 ( K i = 0.04 ± 0.02 nM and 0.07 ± 0.02 nM, respectively), human α7 ( K i = 16 ± 2 nM and 22 ± 3 nM, respectively) and muscle α1β1δg ( K i = 2.5 ± 0.9 nM and 5.7 ± 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human α4β2 (EC 50 = 0.7 ± 0.1 μM) and ganglionic (EC 50 = 0.8 ± 0.09 μM) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 μM). Further, A-85380 can activate (EC 50 = 8.9 ± 1.9 μM) currents through channels formed by injection of the human α7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the α7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (−)-nicotine but less potent than (±)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 ± 0.001 μM which is equipotent to (±)-epibatidine, and 20-fold more potent that (−)-nicotine (EC 50 = 0.04 ± 0.009 μM). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [ 125I]α-BgT sites than [ 3H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the αx4β2 subtype relative to the α7 and α1β1δg nAChRs than (±)-epibatidine.

Effect of chronic administration of dihydropyridine Ca 2+ channel ligands on sufentanil-induced tolerance to μ- and κ-opioid agonists in the guinea pig ileum myenteric plexus

The present investigation was aimed at elucidating if the entry of Ca 2+ plays a role in the development of tolerance to μ- and κ-opioid agonists in the guinea pig ileum myenteric plexus. For this purpose, the influence of the L-type Ca 2+ channel modulators nimodipine (Ca 2+ blocker) and Bay K 8644 (Ca 2+ activator) on the expression of tolerance to the inhibitory effects of μ- and κ-opioid agonists in the ileum of guinea pigs rendered tolerant to sufentanil was investigated. Chronic perfusion of guinea pigs with nimodipine (2 μg/μl/h for 7 days) or Bay K 8644 (0.5 μg/μl/h for 7 days) did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) strip from naive animals. Tolerance to sufentanil (a selective μ-agonist) was induced by s.c. implantation of osmotic minipumps for 7 days, which deliver at 2 μg/μl/h. Control groups received saline. Tolerance to sufentanil as well as to U-50,488H (selective κ-agonist) was observed following chronic treatment with sufentanil and was revealed as a rightward shift of the concentration-response curves. Chronic perfusion of guinea pigs with the Ca 2+ antagonist nimodipine concurrently with chronic sufentanil, markedly blocked the expression of tolerance to sufentanil, as well as the cross-tolerance between sufentanil and U-50,488H. On the contrary, when guinea pigs were perfused with the Ca 2+ agonist Bay K 8644 concurrently with sufentanil, it enhanced the magnitude of tolerance to both sufentanil and U-50,488H. These results suggest that, in guinea pig ileum, chronic exposure to opioids may involve the activation of L-type Ca 2+ channel, which would indicate that intracellular Ca 2+ may be one of the final pathways through which myenteric neurons adapt to the chronic opioid exposure.

11-Oxo-tetrodotoxin and a specifically labelled 3H-Tetrodotoxin

Tetrodotoxin was oxidized to a hydrated aldehyde, 11-oxo-tetrodotoxin, which shares the specificity of tetrodotoxin for the Na + channel of the isolated voltage-clamped frog skeletal muscle fiber, but is four to five times more potent. It binds to the solubilized Na + channel of eel electroplax with a similarly higher potency, because of an equilibrium dissociation constant about 0.25, and a dissociation rate constant 2.4 times slower than those for tetrodotoxin. 11-Oxo-tetrodotoxin can be reduced to regenerate a tetrodotoxin, which is chemically and biologically indistinguishable from the original tetrodotoxin. By reducing with tritiated sodium borohydride, a 3H marker can be inserted regiospecifically to yield 11-[ 3H]-tetrodotoxin. Because it has a defined specific activity of > 2.5 Ci/mmole, and a 3H marker which does not exchange with solvent proton, 11-[ 3H]-tetrodotoxin is an ideal tracer for tetrodotoxin. It may enable studies of problems which require higher signals and/or better stability of the marker than those obtainable from currently available tracer Na +-channel ligands.

Aminooxy-analogues of spermidine: new partial agonists and antagonists at the polyamine site of the rat hippocampal NMDA receptor complex

The amino-1-oxy- and amino-8-oxy-analogues of spermidine (1-O-SPD and 8-O-SPD) were tested in vitro with rat hippocampal membranes as potential modulators of the N-methyl- d-aspartate (NMDA) receptor complex via the polyamine regulatory site. In the presence of 1 μM glutamate and glycine, the binding of the NMDA channel ligand [ 3H]MK-801 was stimulated by 8-O-SPD (EC 50 = 50 μM); 1-O-SPD was without significant influence at concentrations up to 1 mM. Addition of 2 and 4 μM of the polyamine agonist spermine eliminated the stimulatory property of 8-O-SPD, whereas 1-O-SPD was inhibitory under these conditions (IC 50 = 274 and 481 μM, respectively). At higher concentrations of spermine, both compounds were inhibitory. Inhibition of [ 3H]MK-801 binding by the inverse polyamine agonists 1,10-diaminodecane, 1,12-diaminododecane, and arcaine was attenuated by 1 mM 1-O-SPD. The data are compatible with the notion that 8-O-SPD is a partial polyamine agonist and that 1-O-SPD is an antagonist without intrinsic activity.

Inhibitors of cyclic-GMP phosphodiesterase alter excitation of Limulus ventral photoreceptors in Ca(2+)-dependent fashion

We have examined the hypothesis that Ca(2+)-dependent cyclic-GMP metabolism may play a role in visual transduction in Limulus photoreceptors. Although phosphoinositide hydrolysis is central to phototransduction and phosphoinositide-dependent Ca(2+)-mobilization seems to be required for transduction, the subsequent steps leading to ion channel gating (the immediate cause of excitation) are not understood. Channels normally opened in response to light can be opened in excised membrane patches by cGMP but not by Ca2+, suggesting that cGMP acts as a channel ligand in excitation. Using phosphodiesterase inhibitors, we investigated whether changes in cGMP metabolism could affect excitation. We report that zaprinast and IBMX increased the amplitudes and retarded the kinetics of physiological light responses. These effects were maximal for brightest stimuli. The effects were markedly enhanced in low Ca2+ conditions. In contrast, excitation induced by direct IP3-injection and by direct Ca(2+)-injection were inhibited. These observations suggest that PI-induced excitation is dependent on cGMP metabolism in a Ca(2+)-dependent manner, and they support the possibility that transduction involves modification of cGMP metabolism by Ca(2+)-release resulting from phosphoinositide hydrolysis.

Cardiac ATP-sensitive K+ channels: regulation by intracellular nucleotides and K+ channel-opening drugs.

ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells where they regulate cardiac function during cellular metabolic impairment. KATP channels have been implicated in the shortening of the action potential duration and the cellular loss of K+ that occurs during metabolic inhibition. KATP channels have been associated with the cardioprotective mechanism of ischemia-related preconditioning. Intracellular ATP (ATPi) is the main regulator of KATP channels. ATPi has two functions: 1) to close the channel (ligand function) and 2) in the presence of Mg2+, to maintain the activity of KATP channels (presumably through an enzymatic reaction). KATP channel activity is modulated by intracellular nucleoside diphosphates that antagonize the ATPi-induced inhibition of channel opening or induce KATP channels to open. How nucleotides will affect KATP channels depends on the state of the channel. K+ channel-opening drugs are pharmacological agents that enhance KATP channel activity through different mechanisms and have great potential in the management of cardiovascular conditions. KATP channel activity is also modulated by neurohormones. Adenosine, through the activation of a GTP-binding protein, antagonizes the ATPi-induced channel closure. Understanding the molecular mechanisms that underlie KATP channel regulation should prove essential to further define the function of KATP channels and to elucidate the pharmacological regulation of this channel protein. Since the molecular structure of the KATP channel has now become available, it is anticipated that major progress in the KATP channel field will be achieved.

Dihydropyridines and verapamil inhibit voltage-dependent K + current in isolated outer hair cells of the guinea pig

Dihydropyridines and verapamil are widely used as blockers of voltage-dependent Ca ++ channels. In this work we show that these compounds can have a direct blocking action on a class of voltage-activated potassium channels. Voltage-dependent whole-cell currents were recorded from isolated guinea-pig outer hair cells (OHCs) under conditions such that the free Ca ++ concentration in both the internal and external solutions was minimized. A substantial Ca ++-independent K + current was revealed by this procedure. Both conventional K + and Ca ++ channel ligands inhibited this current. The order of potency (in terms of the half inhibitory concentrations (IC 50) of channel inhibitors) was: nimodipine (6 μM) > Bay K 8644 (8 μM) > verapamil (11 μM) >4-aminopyridine (22 μM) > nifedipine (32 μM) > quinine (49 μM) > TEA (10236 μM). Except for verapamil, these channel ligands reduced the size of the K + currents without much alteration of the time course of the currents. In contrast, verapamil caused a more than 10-fold increase in the apparent inactivation rate of the K + currents without significantly altering the activation of the currents. The observation that relatively low concentrations of calcium channel ligands can directly inhibit potassium currents in isolated OHCs indicates that caution should be taken when these pharmacological agents are used as tools for studying cochlear hair cell physiology.

Synthesis of carbon-11-, fluorine-18-, and iodine-125-labeled GABAA-gated chloride ion channel blockers: substituted 5-tert-butyl-2-phenyl-1,3-dithianes and -dithiane oxides.

A series of substituted 5-tert-butyl-2-phenyl-1,3-dithianes and 5-tert-butyl-2-phenyl-1,1,3,3-tetraoxo-1,3-dithianes was synthesized as ligands for the GABAA receptor complex-associated neuronal chloride ion channels. The in vitro binding affinities of these compounds for the GABA-gated chloride ion channel were determined by their ability to compete with [3H]TBOB for binding to rat brain slices. Of the eight compounds tested, trans-5-tert-butyl-2-(4-cyanophenyl)-2-methyl-1,1,3,3-tetraoxo+ ++-1,3-dithiane, 9b, trans-5-tert-butyl-2-(4-fluorophenyl)-1,1,3,3-tetraoxo-1,3-dithian e, 10, and trans-5-tert-butyl-2-(4-iodophenyl)-2-methyl-1,1,3,3-tetraoxo-1,3- dithiane, 11, showed moderately high binding affinities (Ki = 41, 180, and 105 nM, respectively). Four radioligand candidates from this series, 5-tert-butyl-2-(4-cyanophenyl)-2-[11C]methyl-1,3-dithiane, [11C]6, 5-tert-butyl-2-(4-[18F]fluorophenyl)-1,3-dithiane, [18F]7, 5-tert-butyl-2-(4-[18F]-fluorophenyl)-1,1,3,3-tetraoxo-1,3- dithiane, [18F]10, and 5-tert-butyl-2-(4-[125I]iodophenyl)-2-methyl-1,1,3,3- tetraoxo-1,3-dithiane, [125I]11, have been successfully prepared for evaluation as in vivo imaging agents useful for positron emission tomography and single photon emission computed tomography. Preliminary in vivo studies indicate significant uptake into mouse brain for [18F]7, [18F]10, and [125I]11.

Structure modeling of the acetylcholine receptor channel and related ligand gated channels

In this model-building study a model for the pore of the acetylcholine receptor channel is proposed. The pore is formed by five α-helices of the M2 segment where three rings of hydrophilic side chains point into the channel lumen. This model is in agreement with most experimental data like photolabeling, drug affinity studies, single channel conductivity measurements and cryo electron microscopy known about this channel.

Improved micro-perfusion chamber for multiple and rapid solution exchange in adherent single cells.

A new method for fast perfusion of single adherent cells during whole-cell and single-channel patch-clamp measurements is described. The main advantages over previous methods are: (1) the solution surrounding a single cell in a Petri dish (or cover slip) can be exchanged without contamination of the entire cell population, and (2) only small quantities of test solution (50 microliters) are required. The method consists of inserting a patch pipette into a micro-perfusion chamber (MPC) where whole-cell and single-channel currents can be studied. A small volume (2-3 microliters) surrounding adherent single cells in a Petri dish is separated by enclosing it in a ring of Silicone rubber which is then pressed to the bottom of the dish. A drug-containing test solution (50 microliters) is supplied to a funnel at the chamber inflow. The MPC volume isolated from the main solution in the Petri dish is rapidly changed when suction is applied to the chamber outflow. The speed of solution exchange in the MPC (16 +/- 5 ms, n = 12) was estimated by observing changes in the tip potential of 1-M omega patch pipettes during rapid chamber perfusion with solutions of different ionic composition. The seal of the MPC with the surface of a Petri dish was approximated by measuring the electrical resistance between the MPC interior and a reference electrode placed in the Petri dish outside the MPC (range 300-500 M omega). Additionally, a radioactive calcium channel ligand [3H]isradipine was added to the MPC and the appearance of radioactivity in the Petri dish (outside the MPC) subsequently measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential Treatment of Alzheimer Disease Using Cholinergic Channel Activators (ChCAs) with Cognitive Enhancement, Anxiolytic-like, and Cytoprotective Properties

Compounds that activate neuronal nicotinic acetylcholine receptors (nAChRs) may have potential benefit in the treatment of dementia, especially Alzheimer disease (AD). This article summarizes the preclinical pharmacology of ABT-418 [(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole], a novel analog of (-)-nicotine that is being clinically evaluated for the treatment of AD. ABT-418 is a cholinergic channel activator (ChCA) with cognitive enhancement and anxiolytic-like activity possessing a substantially reduced side-effect profile compared to (-)-nicotine [Arneric SP, Sullivan JP, Briggs CA, et al. (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)Isoxazole (ABT-418): A novel cholinergic ligand with cognition enhancing and anxiolytic activity. I. In vitro activity. J Pharmacol Exp Ther 1994 ;270:310-318; Decker MW, Brioni JD, Sullivan JP, et al. (S)-3-Methyl-5-( 1-Methyl-2-Pyrrolidinyl)Isoxazole (ABT-418): A novel cholinergic ligand with cognition-enhancing and anxiolytic activities: II. In vivo characterization. J Pharmacol Exp Ther 1994a;270:319-328; Decker MW, Curzon P, B rioni JD, Arne ric SP. Effects of ABT-418, a novel cholinergic channel ligand, on place learning in septal-lesioned rats. Eur J Pharmacol 1994;261:217-222; Garvey DS, Wasicak JT, Decker MW, et al. Novel isoxazoles which interact with brain cholinergic channel receptors have intrinsic cognitive enhancing and anxiolytic activities. J Med Chem 1994;37:1055-1059]. ABT-418 may be the first agonist of nAChRs to be developed and evaluated specifically for the treatment of AD. Some brief speculation will be given on the potential benefits that this or other ChCAs may have in treating neurodegenerative disorders as compared with (-)-nicotine, and how this differs from other potential treatment approaches.

Role of extracellular Ca2+ in the selective enhancement of contractile responses of arteries from diabetic rats to noradrenaline

Maximum contractile responses of diabetic aortas incubated in the absence of extracellular Ca2+ to increasing Ca2+ (0.01-10 mM) in the presence of 1 microM noradrenaline, but not 40 mM KCl, were significantly increased compared with those of age-matched control rats. Maximum contractile responses of both aortas and mesenteric arteries from diabetic rats to noradrenaline, but not KCl, in the presence of extracellular Ca2+ (2.5 mM) were also significantly enhanced. The Ca2+ channel antagonists verapamil and nifedipine and the Ca2+ channel agonist BAY K8644 produced a similar percentage change in the magnitude of the noradrenaline response in arteries from both control and diabetic rats. These data confirm the selective nature of the enhancement of contractile responses of arteries from diabetic rats to noradrenaline and suggest that this may be mediated in part through enhanced noradrenaline-induced influx of extracellular Ca2+ through channels sensitive to the Ca2+ channel ligands. However, this does not appear to be the only explanation for the enhanced contractile responses of diabetic arteries to noradrenaline, since in the presence of maximum concentrations of nifedipine (3 microM) and verapamil (10 microM), responses of diabetic arteries to noradrenaline were still greater than those of control arteries.

Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx.

Analogs of the seco-steroid hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)-2D3] can preferentially stimulate genomic or nongenomic signaling pathways in osteoblasts. In this study, we used 1,25-(OH)2D3 analogs and voltage-sensitive Ca2+ channel (VSCC) ligands, including dihydropyridines (Bay K 8644 and nitrendipine), in an osteosarcoma cell model to examine the relationship between 1,25-(OH)2D3-stimulated Ca2+ influx and genomic and nongenomic pathways leading to osteoblast activation. Northern blotting experiments demonstrated that an analog of 1,25-(OH)2D3, 1,24-dihydroxy-22-ene-24-cyclopropyl D3, increased messenger RNA (mRNA) levels of both osteopontin (OPN) and osteocalcin (OCN) without triggering Ca2+ influx through VSCCs. Nitrendipine (an inhibitor of L-type VSCCs) did not block the mRNA increase induced by either analog 1,24-dihydroxy-22-ene-24-cyclopropyl D3 or 1,25-(OH)2D3. 1-Deoxy analogs of 1,25-(OH)2D3, 25-hydroxy-16-ene-23-yne-D3, or 25-hydroxy-23-yne-D3, which stimulate Ca2+ influx, did not produce mRNA accumulation for OPN and OCN, consistent with their poor binding to nuclear receptors. Likewise, Bay K 8644, an agonist of VSCCs that produces Ca2+ influx, did not increase mRNA levels for OPN or OCN. Experiments using a construct derived from the sequence of the genomic OPN promoter region and a luciferase reporter confirmed the analog specificity in stimulating transcription. Together these results indicate that 1,25-(OH)2D3-mediated up-regulation of genes encoding OPN and OCN is independent of Ca2+ influx and suggest that the stimulation of Ca2+ influx by 1,25-(OH)2D3 is not required for target gene activation.

Molecular interaction between ryanodine receptor and glycoprotein triadin involves redox cycling of functionally important hyperreactive sulfhydryls

The fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) has been shown to selectively form Michael adducts with hyperreactive sulfhydryls on the skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J.J., Zable, A.C., and Pessah, I.N. (1994) Mol. Pharmacol. 45, 189-200). The present report demonstrates a functionally important interaction between RyR1 and triadin which involves, in part, redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyR1 and triadin only in the presence of Ca2+ channel inhibitors (Mg2+, neomycin, ruthenium red, or anti-triadin antibody). Treatment of SR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics > 10-fold, 2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex (HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyR1 and triadin by Western blot and immunoprecipitation analyses. High-affinity [3H]ryanodine-binding sites are immunoprecipitated by either anti-RyR1 or anti-triadin antibody dose dependently. 1,4-Naphthoquinone (< or = 40 pmol/micrograms protein) selectively oxidizes hyperreactive sulfhydryls on RyR1 and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by beta-mercaptoethanol or dithiothreitol into its component RyR1 and triadin promoters. The results provide direct evidence for the existence of a functionally important complex between RyR1 and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1-triadin complex can signal information across the SR membrane.

Regulation of acetylcholinesterase mRNA stability by calcium during differentiation from myoblasts to myotubes.

The expression of acetylcholinesterase (AChE), nicotinic acetylcholine receptors (nAChR), and their corresponding mRNAs increases dramatically during the conversion of myoblasts to myotubes in C2-C12 cells. The increase in expression of nAChR arises from transcriptional activation of the genes encoding the receptor subunits, whereas stabilization of labile transcripts is primarily responsible for enhanced AChE expression. In a search for the signaling pathways responsible for stabilization of the AChE mRNA, we found that ryanodine, synthetic ryanodine receptor antagonists and L-type, but not N-type, Ca2+ channel blockers inhibit the differentiation-induced expression of AChE mRNA, but not the nAChR mRNA. Selective inhibition of increased expression of AChE is also evident. Inhibition by ryanodine and nifedipine is additive suggesting different target sites for the two Ca2+ channel ligands. Ryanodine binding sites can be detected in both myoblasts and myotubes, but they increase substantially during differentiation. Rates of AChE gene transcription are not altered by ryanodine and nifedipine, indicating that decreased Ca2+ availability prevents stabilization of the mRNA normally seen with differentiation. Muscle cells still undergo elongation and fusion in the presence of ryanodine or L-type Ca2+ channel antagonists. Ryanodine block is fully reversible, indicating functional integrity of the cellular expression system after the drug treatment. These findings indicate that intracellular ryanodine-sensitive calcium channels and extracellular L-type Ca2+ channels link to play an important role in stabilizing AChE mRNA and suggest that transient increases in intracellular Ca2+ may be critical for the commitment of AChE expression during myogenesis.

Heterocyclic amino alcohols related to ifenprodil as σ receptor ligands: binding and conformational analyses

The interaction of a novel series of heterocyclic amino alcohols with the σ receptor site was assessed using radioligand binding and computerized molecular modelling. All heterocyclic amino alcohols, like the structurally related ifenprodil, fully inhibited the specific binding of [ 3H]R(+)-3-(3-hydroxyphenyl)- N-(1-propyl)piperidine ([ 3H]3-PPP) to rat cerebral cortical membranes. All compounds recognised two populations of binding sites labelled by [ 3H]3-PPP and the proportion of sites in the high affinity state was 60–80% of the total sites. Some of the heterocyclic amino alcohols also displayed similar affinity for α 1-adrenoceptors labelled by [ 3H]prazosin, where the pattern of inhibition appears to be stereospecific, unlike that seen with the binding of [ 3H]3-PPP. The amino alcohols had negligible affinity for sites labelled by the N- methyl- D-aspartate channel ligand, [ 3H]-( N-1-[thienyl]cyclohexyl)piperidine. Quantitative conformational analyses indicated that the heterocyclic amino alcohols and ifenprodil fitted well to a σ receptor site model; low energy conformers could be superimposed like other potent σ receptor ligands with confidence to the σ receptor model. Our results define a new class of σ receptor ligands and extend the understanding of the molecular requirements for drugs active at the σ receptor.