Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle

The acetylcholine receptor (AChR) content and the autorphosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rat soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [¹²⁵I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro³²P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the³²P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the³²P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.Abbreviations ACh acetylcholine - AChR acetylcholine receptor(s) - BUTX alpha-bungarotoxin - Kd kilodalton - PAGE polyacrylamide gel electrophoresis - R-II regulatory subunit of cyclic AMP-dependent protein kinase type II - SDS sodium dodecyl sulfate     

Stretch activated ion channels in ventricular myocytes

Patch-clamp recordings from ventricular myocytes of neonatal rats identified ionic channels that open in response to membrane stretch caused by negative pressures (1 to 6 cm Hg) in the electrode. The stretch response, consisting of markedly increased channel opening frequency, was maintained, with some variability, during long (>40 seconds) stretch applications. The channels have a conductance averaging 120 pS in isotonic KCl, have a mean reversal potential 31 mV depolarized from resting membrane potential, and do not require external Ca⁺⁺ for activation. The channels appear to be relatively non-selective for cations. Since they are gated by physiological levels of tension, stretch-activated channels may represent, a cellular control system wherein beat-to-beat tension and/or osmotic balance modulate a portion of membrane conductance.Abbreviations SACs stretch-activated channels - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity

In the present study, culf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl₂. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [³H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5, while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [³H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Stereoselective Nicotine-Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Using a sensitive perfusion system we have studied the nicotine-induced release of [3H]dopamine ([( 3H]DA) from striatal synaptosomes. Nicotine-evoked release was concentration dependent with an EC50 of 3.8 microM. The response to 1 microM nicotine was comparable to that to 16 mM K+; 10 microM veratridine evoked a larger response. All three stimuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 microM (-)-nicotine (100 microliters) at 30-min intervals resulted in similar levels of [3H]DA release; higher concentrations of (-)-nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100-fold higher concentration of (+)-nicotine was necessary to evoke the same level of response as 1 microM (-)-nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high-affinity nicotine binding sites described in mammalian brain.     

Effect of Ethanol Administration on Striatal D1 and D2 Dopamine Receptors

Chronic in vivo exposure of rats to ethanol in a complete liquid diet for 14 or 21 days produced a behavioral tolerance to the acute injection of ethanol. After 21 days, but not 14 days, of chronic exposure, there was a significant increase in the maximum density of striatal D1 and D2 dopamine receptors without a change in these receptors' affinities. A 24-h withdrawal from the 21-day exposure did not alter the observed increase in density. Both the level and duration of ethanol exposure appear to be important variables for demonstration of an increase in striatal D1 and D2 dopamine receptors.     

Kinetic Modulation by GABAergic Agents of High- and Low-Affinity Binding of [3H]Methyl β-Carboline-3-Carboxylate

The kinetics of dissociation of [3H]methyl beta-carboline-3-carboxylate (beta-CCM) binding was studied in a synaptosomal membrane preparation of rat cerebral cortex. Dissociation was biphasic: a faster phase (10-30% contribution) was followed by a slower phase. Picrotoxin pretreatment at 22 degrees C enhanced the equilibrium binding of [3H]beta-CCM. The half-life of the slower phase of beta-CCM dissociation (t1/2II) was increased by 60 muM picrotoxin from 1.7 min to 3.3 min. The dissociation of [3H]beta-CCM was identical when initiated by an excess of either diazepam or beta-CCM. Quasi-equilibrium Scatchard analysis of [3H]beta-CCM binding was performed by a kinetic separation of the rapid and slow phases of dissociation. The slow and rapid phases represented beta-CCM binding sites of high and low affinity, respectively. The dissociation of [3H]beta-CCM (control t1/2II = 2.0 min) was decelerated by the gamma-aminobutyric acid (GABA) antagonist 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135) (t1/2II = 2.5 min) and accelerated by GABA (t1/2II = 1.6 min). GABA inhibited both high- and low-affinity beta-CCM bindings.     

Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N-Methyl-d-Aspartate Receptor Ligands

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20-30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-D-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD = 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki = 31 nM) and the anticonvulsant MK-801 (Ki = 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)