Pharmacology of the neuronal nicotinic acetylcholine receptor of cultured Kenyon cells of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

We investigated the pharmacology of the nicotinic acetylcholine receptor of honeybee Kenyon cells, a subset of olfactory interneurons, which are crucial for olfactory learning and memory. Whole-cell currents were recorded using patch-clamp techniques. Pressure application of agonists induced inward currents in cultured Kenyon cells at holding potentials of –110 mV. Acetylcholine or carbamylcholine were full agonists, nicotine, epibatidine and cytisine were only partial agonists. Coapplications of these partial agonists with acetylcholine reduced the current amplitude. The most efficient antagonists were dihydroxy--erythroidine (EC₅₀=0.5 pmol·l^–1) and methyllycaconitine (EC₅₀=24 pmol·l^–1). The open channel blocker mecamylamine, d-tubocurarine and hexamethonium were rather weak blockers of the honeybee nicotinic response. Bath applications of the muscarinic antagonist atropine inhibited nicotinic currents dependent on concentration (EC₅₀=24.3 mol·l^–1). Muscarine, pilocarpine or oxotremorine (1 mmol·l^–1) did not induce any measurable currents. The non-cholinergic drugs strychnine, bicuculline and picrotoxin partially and reversibly blocked the acetylcholine-induced currents. Our results indicate the expression of only one nicotinic acetylcholine receptor subtype in cultured Kenyon cells. Muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the honeybee nicotinic receptor from the typical nicotinic receptor of vertebrates and from many described insects receptors.     

Respiration of the hydrogenosome-containing fungus Neocallimastix patriciarum

Mass spectrometric determinations of O₂ affinities by the rumen fungus Neocallimastix patriciarum indicated a stable respiration under liquid phase O₂ concentrations up to 10 M, the apparent K _m for O₂ under these conditions was 4.0 M. Exposure to O₂ concentrations in excess of 10 M resulted in rapid inactivation of the observed respiration. Calculated H₂ evolution rates for the organism are 8.1 nmol min^-1 per mg of protein. Exposure to liquid-phase O₂ concentrations in excess of 1.4 M caused 50% inhibition of H₂ production. That superoxide and peroxide are amongst the products of respiration was shown by the use of ESR spectroscopy with the spin trapping agent 5,5-dimethyl-l-pyrroline-N-oxide. An active superoxide dismutase was present, but catalase could not be detected.Abbreviations ESR electron spin resonance - DMPO 5,5-dimethyl-l-pyrroline-N-oxide - DETAPAC diethylene-triamine pentaacetic acid     

Functional and neurochemical evidence that neurotensin-induced release of acetylcholine from Auerbach's plexus of guinea-pig ileum is presynaptically controlled via α2-adrenoceptors

The effect of neurotensin (NT) on [³H]acetylcholine release and contraction from isolated longitudinal muscle strip of guinea-pig ileum was examined. Neurotensin dose-dependently enhanced the release of [³H]-acetylcholine. This effect of neurotensin was inhibited by stimulation of ₂-adrenoceptors: noradrenaline, clonidine, xylazine or dexmedetomidine (₂-adrenoceptor agonists) inhibited neurotensin-induced release of acetycholine (ACh) as well as the contractions, while CH-38083 or yohimbine (₂-adrenoceptor antagonist) prevented this inhibitory effect. Our findings suggest that neurotensin may play a neuromodulatory role in the regulation of cholinergic neuronal activity in the gut and this modulatory effect is continuously controlled by the tonic activity of the sympathetic nervous system: endogenous noradrenaline release is capable of reducing the release of ACh and the consequent contraction of the gut enhanced by neurotensin.     

Converging adrenergic and cholinergic mechanisms in the inhibition of Cl secretion in fish opercular epithelium

Summary The rate of Cl secretion (I _sc) by the opercular epithelium ofFundulus heteroclitus is stimulated by elevations in cyclic AMP (cAMP) levels elicited via ₁-adrenergic receptor activation, and inhibited by both ₂-adrenergic and muscarinic cholinergic receptor activation via mechanisms presently unknown. A comparison of these two inhibitory responses was made using clonidine, an ₂-adrenergic agonist, and acetylcholine (ACh), a cholinergic agonist. The dose required for maximum inhibition was 100 times greater for ACh, but in all other respects the responses elicited by both agonists were statistically indistinguishable. Adrenergic antagonists did not diminish the ACh inhibition, and cholinergic antagonists did not diminish the clonidine inhibition, indicating that the two receptor types were distinct from each other. In control tissues and tissues pretreated with agents that increase cAMP levels (isoproterenol, IBMX, forskolin), both ACh and clonidine had no effects on cyclic AMP levels, indicating an inhibitory mechanism independent of adenylate cyclase. Neither Ca-free media nor a variety of calcium antagonists diminished the ACh or clinidine inhibitions. These results suggest that the ₂-adrenergic and muscarinic cholinergic pathways converge into a common pathway to inhibit Cl secretion by a mechanism not involving adenylate cyclase or the mobilization of either extracellular or intracellular calcium stores.Abbreviations ACh acetylcholine - G ₁ transepithelial conductance - I _sc short circuit current ( chloride secretion) - IBMX 3-isobutyl-1-methyl xanthine     

Decahydroquinoline alkaloids: Noncompetitive blockers for nicotinic acetylcholine receptor-channels in pheochromocytoma cells andTorpedo electroplax

In pheochromocytoma PC12 cells, (+)-cis-decahydroquinoline 195A (5-methyl-2-propyl-cis-decahydroquinoline) and (+)-perhydro-cis-decahydroquinoline 219A (2,5-dipropyl-cis-decahydroquinoline) inhibit carbamylcholine-elicited sodium flux with IC₅₀ values of 1.0 and 1.5 M, respectively. Both of these decahydroquinolines appear to enhance desensitization, although apparent lack of complete removal of (+)-perhydro-cis-219A by washing complicates interpretation of the effects of that agent. A series of cis- and trans-decahydroquinolines with substituents in the 2- and 5-position also exhibit structure-dependent inhibition of carbamylcholine-elicited sodium flux in PC12 cells and all of the decahydroquinolines inhibit binding of the noncompetitive blocking agent [³H]perhydrohistrionicotoxin to muscle-type nicotinic acetylcholine receptor-channels in membranes fromTorpedo electroplax. The K_i values in electroplax membranes range from 1.4 to 7.9 M, making these alkaloids comparable in potencies to the histrionicotoxins. Potencies are increased 2- to 3-fold in the presence of an agonist, carbamylcholine. The profile of activities are similar in PC12 cells and electroplax membranes. The cis- and trans-decahydroquinolines represent another class of noncompetitive blockers for acetylcholine receptor-channels with similar activity for both muscle-type and ganglionic type nicotinic receptors.     

Nicotinic receptor-elicited sodium flux in rat pheochromocytoma PC12 cells: Effects of agonists,antagonists, and noncompetitive blockers

Nicotinic agonists stimulate²²Na flux in rat pheochromocytoma PC12 cells. The stimulatory effect of carbamylcholine is maximal at 1 mM, while the stimulatory effect of nicotine and anatoxin maximize at the same level at 100 M and 10 M, respectively. The tertiary amines arecolone and isoarecolone have no effect on flux at 100 M, while the methiodides at 100 M stimulate flux to an extent similar to 1 mM carbamylcholine. Dihydro and alcohol analogues of isoarecolone methiodide have markedly smaller effects on flux. A preincubation for 2 to 20 min with carbamylcholine (2 mM), nicotine (300 M), anatoxin (30 M) or isoarecolone methiodide (100 M) causes marked desensitization to a subsequent carbamylcholine-elicited stimulation of flux. d-Tubocurarine, mecamylamine, hexamethonium, and chlorisondamine inhibit carbamylcholine-elicited flux with IC₅₀ values of 1.0, 0.8, 43, and 0.020 M, respectively. Atropine has no effect at 1 M, but reduces the response to carbamylcholine by 50% at 8.6 M, presumably as a noncompetitive blocker. Other noncompetitive blockers of nicotinic acetylcholine-receptors, such as histrionicotoxins, gephyrotoxin, pumiliotoxin C, phencyclidine, bupivacaine and piperocaine, inhibit carbamylcholine-elicited stimulation of²²Na flux with IC₅₀ values from 0.3 to 1.8 M. In contrast to d-tubocurarine, which inhibits carbamylcholine-elicited desensitization, and mecamylamine, which has no apparent effect on desensitization, chlorisondamine and certain noncompetitive blockers appear to enhance desensitization. The effects of agonists, antagonists and noncompetitive blockers at the neuronal nicotinic acetylcholine receptor-channel of PC12 cells are compared to their effects on binding of [¹²⁵I]-bungarotoxin to agonist-recognition sites and of [³H]perhydrohistrionicotoxin to noncompetitive blocker sites of the nicotinic acetylcholine receptor-channel of electric ray (Torpedo) electroplax membranes. There are marked differences in relative potencies for the two types of nicotinic acetylcholine receptor-channel.     

Intracellular calcium mobilization on stimulation of the muscarinic cholinergic receptor in chick limb bud cells

Summary Cell suspensions of chick limb buds (stage 23/24) were loaded with the fluorescent Ca²⁺ chelator chlorotetracycline. Fluorescence was monitored in a spectrofluorometer. Stimulation with acetylcholine induced a fluorescence decrease, indicating intracellular Ca²⁺ mobilization. The fluorescence decrease triggered by acetylcholine was inhibited by muscarinic but not by nicotinic antagonists, indicating that a muscarinic acetylcholine receptor is involved. The muscarinic receptor in the chick limb bud has a characteristic pharmacological profile: acetylcholine, carbachol and acetyl--methylcholine functioned as full agonists triggering maximal fluorescence decrease. Bethanechol was less effective, producing only one-third of the maximum response. Pilocarpine and oxotremorine, two classical agonists in other systems, were ineffective and functioned as antagonists. In the chick limb bud, cholinesterase, choline acetyltransferase and the presence of a muscarinic receptor have been demonstrated in previous studies. The present experiments show that stimulation of the embryonic muscarinic receptor leads to intracellular Ca²⁺ mobilization.     

M3 muscarinic receptors on murine HSDM1C1 cells: Further functional,regulatory, and receptor binding studies

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M₃-selective radioligand, [³H]4-DAMP, to cell homogenates was characterized. Carbachol (EC₅₀=9 M), (+)muscarine (EC₅₀=4.5 M) and cis-dioxolane (EC₅=0.72 M) were full agonists which stimulated PI turnover by 13.3±1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM–10 M muscarine during the last 24h of [³H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cisdioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. pertussis toxin (5–2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 M) PI turnover. The half-maximal effect of TPA was produced at 1.8±0.3 nM. [³H]4-DAMP binding to cell homogenates was of high affinity (K_d=0.19±0.04 nM) and moderate capacity (B_max=201±22 fmol/mg protein). The pharmacological specificity (4-DAMP>p-FHHSiD>dicyclomine>pirenzepine>methoctramine>AFDX-116 >gallamine) resembled that for [³H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M₃ receptors on HSDM1C1 cells. These receptors have been shown to be influenced by pertussis toxin, an active phorbol ester and to exhibit desensitization.     

Does acetylcholine change the electrical resistance of the basal membrane of secretory cells in eccrine sweat glands?

Summary The present experiment was intended to study whether or not acetylcholine decreases the electrical resistance of the basal membrane of secretory cells in stimulating eccrine secretion of fluid and electrolytes. An isolated segment of the secretory coil of the monkey palm eccrine sweat gland was dissected outin vitro and immobilized in the tip of a constriction pipette. Using a bridge-balanced single glass microelectrode, input impedance of the secretory cell was compared before and after local superfusion of acetylcholine in each cell. The mean input impedance was 27m, which did not significantly change after application of acetylcholine. Between 15 and 30 sec after cessation of acetylcholine superfusion, input impedance increased by 42% and then returned to normal within 60 sec. The current-induced voltage deflection due to intraluminally injected current pulse was measured across both the basal membrane (V _b ) and the epithelial wall (V _t ) as qualitative measures of the respective membrane resistances. Both V _b and V _t increased by about 10%, but their ratio remained unchanged after stimulation with acetylcholine. A Ca⁺⁺ ionophore, A23187, which is as potent a stimulant of eccrine sweat secretion as acetylcholinein vitro, also failed to change the above two parameters. It was concluded that the decrease in the electrical resistance of the basal membrane of the secretory cells could not be detected in the sweat gland after stimulation with acetylcholine or A23187. The possibility was discussed that the action of acetylcholine at the basal membrane is one of enhancing the activity of the nonconductive pathway rather than the conductive pathway in this exocrine gland.     

Functional Neurochemical Evidence for the Presence of Presynaptic Nicotinic Acetylcholine Receptors at the Terminal Region of Myenteric Motoneurons: A Study with Epibatidine

The aim of this study was to verify the presence of presynaptic nicotinic acetylcholine receptors (nAChRs) at the terminals of myenteric motoneurons using a potent and highly selective nicotinic agonist, epibatidine. We examined contraction, and release of [³H]ACh on a guinea-pig longitudinal muscle strip preparation. First, we compared the ability of epibatidine and nicotine to induce isometric contraction and found epibatidine (EC₅₀ = 23.1 nM) to be 300-fold more potent than nicotine (EC₅₀ = 7.09 M). The release and contraction induced by 30 nM epibatidine were inhibited by the nicotinic antagonist mecamylamine (3 M) and the Na1-channel blocker TTX (1 M), indicating that the effects are mediated via nAChRs and are fully dependent on the propagation of action potentials. Atropine (0.1 M) significantly increased the [³H]ACh release but could not block contraction suggesting that a substantial part of the response develops via a noncholinergic mechanism. Epibatidine at a higher concentration (300 nM) induced contraction, which was only partly (45%) inhibited by TTX (1 M). The TTX-resistant contraction, however, was completely blocked by mecamylamine (3 M). Our data provide functional neurochemical evidence for the existence of presynaptic nAChRs at myenteric motoneuron terminals and suggest that these receptors can be activated only/by a higher concentration of agonists.     

Chloride secretion by canine tracheal epithelium: I. Role of intracellular cAMP levels

Summary We measured the short-circuit current (I _sc) across canine tracheal epithelium and the intracellular cAMP levels of the surface epithelial cells in the same tissues to assess the role of cAMP as a mediator of electrogenic Cl secretion. Secretogogues fall into three classes: (i) epinephrine, prostaglandin (PG) E₁, and theophylline increase bothI _sc and cellular cAMP levels; (ii) PGF₂ and calcium ionophore A23187, increaseI _sc without affecting cell cAMP levels at the doses employed; and (iii) acetylcholine, histamine, and phenylephrine do not alter eitherI _sc or cAMP levels.These findings indicate that: (i) increases in cAMP or Ca activity stimulate electrogenic Cl secretion by the columnar cells of the surface epithelium; (ii) cAMP mediates the effects of PGE₁ and -adrenergic agonists; (iii) a strict correlation between cAMP levels and Cl secretion rate is not apparent from spontaneous variations in these parameters or from dose-response relations ofI _sc and cAMP to epinephrine concentration; and (iv) acetylcholine, histamine, and phenylephrine, agents that stimulate electrically-neutral NaCl secretion by submucosal glands, do not evoke cAMP-mediated, responses by the surface epithelium.Addition of 10^–6 m indomethacin (or other prostaglandin synthesis inhibitors) to the mucosal solution decreasesI _sc and cellular cAMP levels and reduces the release of PGE₂ into the bathing media by 80%. Indomethacin does not interfere with the subsequent secretory response to PGE₁. This suggests that endogenous prostaglandin production underlies the spontaneous secretion of Cl across canine tracheal epithelium under basal conditions.     

5,8-Disubstituted indolizidines: A new class of noncompetitive blockers for nicotinic receptor-channels

A series of 8-methyl-5-substituted indolizidines inhibit binding of the noncompetitive blocking agent [³H]perhydrohistrionicotoxin to muscle-type nicotinic acetylcholine receptor-channels in membranes fromTorpedo electroplax. The K_i values range from 0.16 to 1.12 M, making these alkaloids among the most potent ligands for this site. Unlike most noncompetitive blockers, the potencies of the 8-methyl-5-substituted indolizidines arereduced in the presence of carbamylcholine. Indolizidine 205A (8-methyl-5-(4-pentynyl)indolizidine) is unique in enhancing binding of [³H]perhydrohistrionicotoxin by 1.5-fold. The enhancement is at a maximum at 0.01 to 0.1 M, followed by progressive inhibition with an IC₅₀ of about 20 M. In the presence of carbamylcholine, which itself enhances binding of [³H]perhydrohistrionicotoxin, indolizidine 205A causes only an inhibition of binding with an IC₅₀ of about 10 M. Indolizidines with a hydroxy substituent on the 8-methyl group have very low activity. None of the indolizidines affect binding of [¹²⁵I]-bungarotoxin to acetylcholine recognition sites. In pheochromocytoma PC12 cells, indolizidine 205A has no agonist activity, but only inhibits carbamylcholine-elicited²²Na⁺ influx. The profile of potencies for the 8-methyl-5-substituted indolizidines is similar in electroplax membranes and PC12 cells. Indolizidines 205A and 209B (8-methyl-5-pentylindolizidine) have no apparent effect on desensitization of receptors in PC12 cells. The 5,8-disubstituted indolizidines appear to represent an atypical and potent class of noncompetitive blockers for muscle-type and ganglionic nicotinic receptor-channels.     

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

Summary Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits⁸⁶Rb flux (IC₅₀=6.6m), and [³H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC₅₀=17 m and 0.22 m, respectively). From an indirect Hill plot of the inhibition of [³H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in -bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.     

Possible modulation of phosphorylation of acetylcholine receptor-enriched membrane preparations

During phosphorylation of acetylcholine receptor (AChR)-enriched membrane preparations fromTorpedo fuscomaculata, phosphate is incorporated into a single protein, with a molecular weight corresponding to that of one of the receptor subunits (37,000 daltons). This protein also seems to contain the receptor binding site. ATP binds to four protein species, one of which corresponds to a different subunit of the receptor (molecular weight 45,000). Phosphorylation of these membrane preparations is affected by several factors, known to be involved in postsynaptic events. Ca²⁺ (10 M) inhibits the reaction, whereas cGMP (20 M), causes stimulation. Furthermore it has been shown that the agonists, acetylcholine, and carbamylcholine (10 M and 1 M) stimulate the phosphorylation reaction, while the antagonists, tubocurarine, hexamethonium, and decamethonium (1 M), cause inhibition.     

Conformation of acetylcholine receptor in the presence of agonists and antagonists

The conformations of acetylcholine receptor fromTorpedo californica in the absence and presence of agonists, antagonists, and local anesthetics were studied by circular dichroism (CD). Without ligands, the receptor had about 40% helix, 20% -sheets, and 10% -turns as analyzed from its far-UV CD spectrum. Its near-UV CD spectrum resembled that of acetylcholinesterase from the same source. None of the ligands studied altered the far-UV spectrum of the receptor. However, in the near-UV region, carbamylcholine and acetylcholine shifted the Phe and Tyr bands of AChR to less negative, whereas hexamethonium changed the Tyr bands to more negative, indicating that the site of binding of agonists and antagonists and their effect on the conformation of the receptor may be different. Decamethonium, procaine, and lidocaine had no effect on both the far- and near-UV CD spectra of acetylcholine receptor.     

Fluorescent-Labeled Lipophilic Analogues of Serotonin,Dopamine, and Acetylcholine: Synthesis,Mass Spectrometry,and Biological Activity

4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl derivatives of serotonin, dopamine, choline, and N,N-dimethylaminoethanol, with the fluorescence maximum at 512 nm (_exc 470 nm), and 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl derivatives of choline and N,N-dimethylaminoethanol, with the fluorescence maximum at 554 nm (_exc 470 nm), were synthesized. These compounds yield protonated molecular ions of 100% intensity upon mass spectrometry with electrospray ionization at atmospheric pressure. The fragmentation of molecular ions under the conditions of secondary ion mass spectrometry mainly proceeds through the elimination of hydrogen fluoride from the fluorescent core of the molecules. Experiments on sea urchin Lytechinus variegatus embryos and larvae showed that these compounds easily penetrate into the cells and are accumulated in the cytoplasm. They do not differ in their biological activity from similar derivatives of arachidonic acid described previously and are agonists of serotonin or acetylcholine or antagonists of nicotinic acetylcholine receptors.     

Differential Effect of Nicotinic Agonists on the [3H]Norepinephrine Release from Rat Hippocampal Slices

The aim of this study was to investigate the mechanisms involved in the effect of nicotinic agonists on the [³H]norepinephrine ([³H]NE) release from rat hippocampal slices. The stimulatory effect of nicotine, cytisine, epibatidine and anatoxin-A was completely blocked by the nicotinic antagonist mecamylamine (10 M). In contrast, the effect of dimethylphenylpiperazinium (DMPP) was only partially inhibited by mecamylamine but was completely blocked by the NE uptake inhibitor desipramine (DMI, 10 M). Finally, the effect of lobeline was not affected by mecamylamine and was only partially blocked by DMI. Our data indicate that the majority of nicotinic agonists increase the release of [³H]NE exclusively via stimulation of nicotinic acetylcholine receptors (nAChRs). DMPP, in addition to the stimulation of nAChRs, also evokes a carrier-mediated release. Lobeline has no stimulatory effect on nAChRs, induces a carrier-mediated release and has a further action of unidentified mechanism. Our results suggest that special caution is required for the interpretation of data, when DMPP or lobeline are used as nicotinic agonists.     

Effect of amitraz on contractions of the guinea-pig ileum in vitro

• 1.1. The effect of the formamidine pesticide amitraz on the motility of isolated pieces of guinea-pig ileum was studied.
• 2.2. Contractions of the ileum stimulated by histamine and the histamine H₁ agonists 2-methylhistamine and 2-pyridylethylamine were inhibited by amitraz at a concentration of 1 μg/ml.
• 3.3. Amitraz did not inhibit contractions stimulated by acetylcholine, methacholine or dimethylphenylpiperazinium.

4. When pieces of intestine were left exposed to amitraz for several minutes they began to contract. These contractions appeared as rhythmic contractions and their strength did not appear to be increased by increasing the concentration of amitraz.5. It is concluded that amitraz inhibits the action of histamine H₁ agonists on the guinea-pig ileum and itself has a weak agonist effect.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

Carbamylcholine and acetylcholine-sensitive,cation-selective ionophore as part of the purified acetylcholine receptor

Summary Black lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric rayTorpedo californica or its tryptic digest. In both cases, conductance of cations increased and was dependent on the concentration of the receptor protein. Conductance of Ca⁺⁺ was dependent on its concentration, but addition of carbamylcholine gave no reproducible or consistent effects. Only in the case of the tryptic digest of the acetylcholine receptor did carbamylcholine and acetylcholine consistently induce monovalent cation selective conductance (P _{Na, K}P _Cl=4.4). The induced monovalent cationic conductance due to carbamylcholine (10 m) varied from 10- to over 100-fold. Curare (10 m) prevented the action of carbamylcholine.Na-dodecyl sulfate gel electrophoresis of the acetylcholine receptor, before and after tryptic digestion, indicated that this mild enzyme treatment hydrolyzed the receptor molecule subunits. Nevertheless, the receptor molecule retained its full binding of [acetyl-³H]acetylcholine; and analytical gel electrophoresis indicated that it remained intact possibly through hydrogen, hydrophobic and disulfide bonding.