Cholinergic Sites Research Articles

Synthesis and characterization of luminescent cadmium selenide/zinc selenide/zinc sulfide cholinomimetic quantum dots

Luminescent quantum dots conjugated with highly selective molecular recognition ligands are widely used for targeting and imaging biological structures. In this paper, water soluble cholinomimetic cadmium selenide (core), zinc selenide/zinc sulfide (shell) quantum dots were synthesized for targeting cholinergic sites. Cholinomimetic specificity was incorporated by conjugation of the quantum dots to an aminated analogue of hemicholinium-15, a well known competitive inhibitor of the high affinity choline uptake transporter. Detailed evaluation of the nanocrystal synthesis and characterization of the final product was conducted by (1)H and (31)P NMR, absorption and emission spectroscopy, as well as transmission electron microscopy.

Dual effect of lobeline on α4β2 rat neuronal nicotinic receptors

The effect of lobeline on rat α4β2 nicotinic receptors expressed in COS cells was studied using the patch-clamp technique. Currents were recorded in whole-cell mode 2–4 days after cell transfection by plasmids coding the α4β2 combination of receptor subunits. In cells sensitive to acetylcholine, the application of lobeline evoked minor responses (up to 2% of maximal acetylcholine response). When acetylcholine was applied to the background of an already running application of lobeline, acetylcholine responses were inhibited in a concentration- and time dependent manner. However, when lobeline was applied simultaneously with acetylcholine without any prepulse or during an already running application of acetylcholine, the acetylcholine responses were potentiated up to 300–600% of that of the control. The site of lobeline action overlaps with the cholinergic site, as was proven by the partially protective effect of (+)-tubocurarine. Thus, lobeline can apparently desensitize receptors when applied alone (inhibition) whereas its binding to a second agonist site with the first one already occupied by acetylcholine leads to channel opening (potentiation).

Sevoflurane Anesthesia Alters Exploratory and Anxiety-like Behavior in Mice Lacking the β2Nicotinic Acetylcholine Receptor Subunit

Preexisting cognitive impairment and advanced age are factors that increase the risk of developing postoperative cognitive dysfunction. Because anesthetic agents interfere with cholinergic transmission and as impairment of cholinergic function is associated with cognitive decline, the authors studied how the volatile anesthetic sevoflurane affects exploratory and anxiety-like behavior in young and aged animals with a genetically modified cholinergic system. Young and aged wild-type and mutant mice lacking the beta2 subunit of the nicotinic cholinergic receptor (beta2KO) were anesthetized for 2 h with 2.6% sevoflurane in oxygen and compared with nonanesthetized controls. Locomotor activity and organization of movement in the open field model were assessed before and 24 h after anesthesia. Locomotor activity and anxiety-like behavior in the elevated plus maze were assessed 24 h after anesthesia. High- and low-affinity nicotinic receptor and cholinergic uptake site densities were evaluated in the hippocampus, amygdala, and forebrain regions using receptor autoradiography. Sevoflurane anesthesia significantly reduced locomotor activity, altered temporospatial organization of trajectories, and increased anxiety-like behavior in young beta2KO mice, whereas no such changes were observed in young wild-type mice. Aged wild-type and beta2KO mice displayed reactions that were similar, but not identical, to the reactions of young mice to sevoflurane anesthesia. However, behavioral changes were not associated with differences in nicotinic receptor or cholinergic uptake site densities. In conclusion, sevoflurane anesthesia altered exploratory and anxiety-like behavior in mice lacking the beta2 nicotinic acetylcholine receptor subunit.

Influence of beta-amyloid peptide on cell membrane lipids and cholinergic receptors in human neuroblastoma SH-SY5Y cells

To study the effects of beta-amyloid peptide (Abeta) on cell membrane lipids and cholinergic receptors of human neuroblastoma cells. Human SH-SY5Y neuroblastoma cells were treated with different concentrations of Abeta(1-42) with and without pretreatment of vitamin E. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction, lipid peroxidation, protein oxidation and phospholipids were measured by spectrophotometry. Levels of cholesterol and unbiquinone were determined by high-performance liquid chromatography (HPLC). The numbers of cholinergic receptor binding sites were determined by receptor binding assay and the protein levels of nicotinic receptor alpha3 and alpha7 subunits were studied by Western blotting. SH-SY5Y cells showed decreased reduction rates of MMT and phospholipids, and increased lipid peroxidation and protein oxidation after exposure to Abeta (0.1 micromol/L) as compared to the control. The number of cholinergic receptor binding sites, the protein level of nicotinic receptor alpha3 and alpha7 subunits and the content of ubiquinone decreased in cells treated with high dose of Abeta (1 micromol/L). Although the level of cholesterol was not changed in any way, vitamin E partially prevented the neurotoxic effects of Abeta. beta-amyloid peptide reduces the level of cell membrane lipids and cholinergic receptors in human SH-SY5Y neuroblastoma cells, likely through the induction of an enhanced oxidative stress.

Role of Cholinergic Mossy Fibers in Vestibular Nuclei in the Development of Vestibular Compensation

Several lines of evidence have suggested that acetylcholine is a possible neurotransmitter/neuromodulator involved in vestibular compensation. However, details of cholinergic effective sites during vestibular compensation remain unclear. In this study, we selectively damaged the rat vestibulo-floccular cholinergic mossy fibers using ethylcholine mustard aziridinium ion. In these animals, unilateral labyrinthectomy caused more severe vestibulo-ocular deficits, especially in the initial stage. These findings suggest that the vestibulo-floccular cholinergic mossy fibers serve to restore the balance between intervestibular nuclear activities in order to induce vestibular compensation in the initial stage.

Cellular localization of estrogen receptors on neurones in various regions of cultured rat CNS: coexistence with cholinergic and galanin receptors

Autoradiographic studies have shown that many neurones in explant cultures of rat neocortex, hippocampus, preoptic area and spinal cord express binding sites for [3H]-estradiol which are distributed over the cell bodies and primary processes. By means of immunohistochemistry, it was observed that neurones were labelled by monoclonal antibodies against estrogen α-receptors and a polyclonal antibody against estrogen β-receptors. Immunoreactivity was distributed over the soma and primary processes of the cells, the nuclei being more intensely stained. Double-immunostaining revealed a colocalization of estrogen α- and β-receptors on approximately half of the neurones in cultures from neocortex and hippocampus whereas in cultures from preoptic area and spinal cord only few cells were double-stained. On many neurones, a coexistence of estrogen receptors and cholinergic muscarinic or nicotinic sites was found. Furthermore, combined autoradiographic and immunohistochemical studies have shown a colocalization of receptors for estrogen and the neuropeptide [125I]-galanin. The coexistence of estrogen and cholinergic sites as well as of estrogen and galanin receptors on the same neurones are discussed with respect to neurodegenerative events such as Alzheimer's disease.

6-Hydroxycatecholine, a Choline-Mimicking Analogue of the Selective Neurotoxin, 6-Hydroxydopamine

The synthesis and properties of 6-hydroxy-N,N-dimethylepinephrine (6-hydroxy-catecholine) are reported. This agent is intended for use as a selective presynaptic cholinotoxin and is based on previously reported neurotoxins of the same type. 6-hydroxycatecholine is a close structural analogue of the catecholaminergic neurotoxin 6-hydroxydopamine, and is expected both to be selectively reactive at cholinergic sites and to undergo less vigorous and potentially more selective inactivating reactions. It is also possible that in specific dementia-inducing pathologies, 6-hydroxycatecholine could be formed endogenously.

Design and stereoselective synthesis of conformationally constrained analogues of Zimeldine

Most of the commonly used antidepressant drug (e. g. imipramine and amitriptyline) have cardiovascular side effects (orthostatic hypotension, tachycardia, arrhythmia) due to the action of these drugs at cholinergic and adrenergic sites (Ankier, 1986). The goal of this research program is to design and sythesize very selective inhibitors for the uptake of either, but not both, norepinephrine (NE) or serotonin (5HT). Examining the relation between the conformation of these molecules and their selectivity for inhibition of the uptake of NE or 5HT could provide an understanding of the molecular mechanisms involved. Such information could lead to even safer antidepressant drugs with diminished cardiovascular side effects. Furthermore, drugs that are selective for inhibition of the uptake of 5HT (e.g. fluoxetine) show promise as aspects for obesity control (Grunewald et al., 1986a). In order to find new drugs with enhanced selectivity for the uptake of 5HT which could be useful as antidepressants requires an understanding of the structural features of these drugs that results in their selectivity for the 5HT uptake carrier. As part of a search for the explanation of the dramatic pharmacological difference between (Z)-Zimeldine (1, a selective inhibitor of the reuptake of serotonin) and (E)-Zimeldine (2, a selective inhibitor of norepinephrine) in presynaptic nerve endings (Hoberg et al., 1981), we sought a convenient synthesis of conformationally defined analogues of 1 and 2 in the quinoline and naphthalene skeleton. We wondered if subtle changes in low energy conformations of the

Cholinergic and purinergic signalling in outer hair cells of mammalian cochlea

Outer (OHC) and inner (IHC) hair cells in the organ of Corti of the mammalian cochlea process sound. OHC and their efferent synapse are part of a feedback system assumed to control and modulate information carried by afferent neurons passing from IHC to the brain. Underlying mechanisms are not well understood. This paper discusses recent progress. In vivo and in vitro information is presented on structure, pharmacology, function and localization of the pre- and postsynaptic acetylcholine receptors (AChRs) at the efferent synapse. Recent data are given on a presynaptic M3 AChR subtype, probably an autoreceptor involved in transmitter release. Data from our lab on specific binding of [ 3H]3-quinuclidinyl benzilate ([ 3H]3-QNB) to non-enzymatically isolated guinea pig OHC reveal a K D several 100 x higher than that for any known muscarinic receptor subtype, including the above-mentioned presynaptic muscarinic AChR of the OHC efferent synapse. The extremely high concentrations of [ 3H]3-QNB needed for any binding at all to OHC thus rule out presynaptic membrane impurities as the cause of such binding, and also the presence of a typical mAChR subtype on OHC. The number of [ 3H]3-QNB binding sites (∼ 10 6/OHC) we found on OHC was 1 10 th of that we found for binding of nicotinic ligands to OHC, further making it questionable that an ACh-binding site on OHC binds [ 3H]3-QNB. Observations may instead point to the possibility of another binding site, e.g. an (allosteric) site involved with the as yet not understood ‘weak’ muscarinic properties of the OHC AChR. Further new data on the OHC AChR confirm reversible α-bungarotoxin, nicotine and d-tubocurarine binding. [ 3H]α-Bungarotoxin and [ 3H]-nicotine binding sites are estimated at ∼6 · 10 7 sites/OHC. Strychnine, a glycine receptor blocker suggested to interfere with cholinergic sites of the efferent OHC synapse, was found to bind to OHC (cold strychnine for unspecific binding). This binding, not seen in the presence of high [glycine], increased in the presence of depolarizing [K +], while ACh (100 μM) had no significant effect. Results suggest strychnine binding to the outside of OHC, but also to sites accessible only after cell depolarization, possibly to the hyperpolarizing Ca 2+-dependent K + channel. Recent molecular cloning of the OHC AChR indicates a novel α-subunit. An often observed ACh-activated Ca 2+-influx close to zero into OHC leaves an unanswered question. OHC also carry P 2-purinergic receptors (P 2Rs), a more rapid ionotropic P 2zR-like subtype and a quantitatively dominating slow metabotropic P 2yR subtype coupled to a G protein-phospholipase C cascade and not desensitized. Both contribute to increased cytoplasmic [Ca 2+], from respectively external and internal sources. Whether or not such receptors are part of efferent synaptic activity is unknown; their localization on the OHC plasma membrane is so far only indirect and synaptic vesicles of the efferent nerve endings have not yet been analyzed for their ATP content. Localization, function and interaction of [Ca 2+] increases triggered by, respectively, ATP and ACh are currently studied in this laboratory.

Prejunctional and postjunctional inhibition of adrenergic transmission in the rat-isolated anococcygeus muscle by cimetidine.

1. Cimetidine and ranitidine can inhibit various cholinergic sites, which can account for some of their clinically documented adverse effects; ranitidine can also inhibit adrenergic transmission, closely resembling the action of guanethidine. The effects of cimetidine on adrenergic transmission in the rat isolated anococcygeus muscle (Acm) were therefore investigated. 2. The contractile (motor) responses of the Acm to electrical field stimulation (EFS; 20-30V, 10 s, 1 ms pulse width, every 2 min) at varying frequencies (Hz: 5, 10, 20) and to 3 microM noradrenaline (NA) were inhibited in a concentration-dependent manner by cimetidine (mM: 1, 2, 4, 8). Inhibition of the EFS-induced responses was inversely related to the stimulation frequency. 3. Cimetidine (nM: 2, 4, 8) produced a concentration-dependent and non-parallel shift of the NA cumulative log concentration-response curves (CRCs; curves 2, 3, 4) to the right of the control curve (curve 1); at the highest concentration (8 mM) used, cimetidine produced a 4.3-fold shift of curve 4 accompanied by a decline of 9.4 +/- 1.5% in the maximal response to NA (compared to essentially no change in maximal responses for the corresponding CRCs in the NA control series). Cimetidine therefore inhibited the postjunctional alpha-adrenoceptor sites. 4. The contractile responses of the Acm to EFS (i.e. prejunctionally mediated responses) were more sensitive to inhibition by cimetidine than the NA-induced (postjunctionally mediated) responses: 8 mM cimetidine inhibited the responses to EFS by about 97%, whereas the responses to NA were inhibited by only 41 +/- 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and ligand specificity of acetylcholinesterase and the nicotinic receptor: a tale of two cholinergic sites.

The functional design of the nAChR and AChE rather than their recognition capacities requires divergence in structure of the two binding sites. The receptor requires co-operativity to link ligand occupation to the response, rapid conformational transitions of activation, and slower transitions of desensitization. Hence, its binding sites have evolved at subunit interfaces. By contrast, AChE functions with a large kcat and a comparatively large Km. To do so, it must force acetylcholine through a low-energy transition site that features tetrahedral rather than the ground-state, trigonal conformation around the carbonyl carbon. This requires a high affinity (KD approximately 10(-17) M) for the enzyme complex of the transient transition state. Interestingly, the three-finger peptide toxins (alpha-bungarotoxin and fasciculin), though closely homologous, use different interaction sites on the receptor (the agonist recognition site) and AChE (a peripheral site). Finally, although the two proteins show co-ordinated expression during muscle differentiation, the receptor relies primarily on transcriptional control while AChE expression is post-transcriptional, being controlled by mRNA stability.

Dual action of ranitidine at cholinergic sites in the rat isolated anococcygeus muscle.

1. The effects of ranitidine on contractile responses of the rat isolated anococcygeus muscle to acetylcholine (ACh) and carbachol (CCh) were investigated. 2. Four consecutive cumulative concentration-response curves (CRCs) for ACh or CCh were constructed in the absence or presence of ranitidine. The mean EC50 values were determined from the individual CRCs and used for the calculation of the corresponding EC50 ratios. 3. Ranitidine (0.01, 0.05 and 0.5 mM) produced a progressively leftward shift of the ACh curves; the corresponding EC50 ratios obtained were 3.2 +/- 0.4, 5.7 +/- 0.5 and 9.4 +/- 1.4. Neostigmine 0.5 microM alone or in combination with ranitidine 0.1 mM produced very marked leftward shifts of the ACh curves (EC50 ratios 68.2 +/- 14.3 and 56.3 +/- 9.6 respectively). In contrast, ranitidine (0.05, 0.5 and 2 mM) produced a rightward and parallel shift of the CCh curves (EC50 ratios 1.0 +/- 0.1, 3.4 +/- 0.32 and 12.5 +/- 1.7 respectively). 4. Ranitidine can therefore mediate a dual action at cholinergic sites in the rat isolated anococcygeus muscle, actions which can be attributed to its inherent anticholinesterase and antimuscarinic activity. The relatively marked anticholinesterase action of ranitidine was rather surprising in view of the limited cholinergic innervation present in the rat anococcygeus muscle.

Affinity ligands and related agents for brain muscarinic and nicotinic cholinergic receptors

This study describes the chemical synthesis and receptor binding characteristics of various affinity ligands and related ligands for brain muscarinic and nicotinic cholinergic receptors, including the 4-bromoacetamidobenzoic acid esters of dimethylaminoethanol (DMBAB) and choline (BABC) and 4-iodoacetamidobenzoylcholine (IABC). The reversible binding of [ 3H]3-quinuclidinylbenzilate ([ 3h]QNB) to calf brain membranes was inhibited in a concentration-dependent and saturable manner by DMBAB, BABC, and IABC with K i , values of 8 × 10 −7, 3 × 10 −7 and 8 × 10 −7M, respectively; the K i , values for inhibition of reversible binding of the nicotinic ligand,[ 3H]methylcarbamylcholine ([ 3H]-MCC), were 1 × 10 −6, 6 × 10 −8, and 1 × 10 −6M, respectively. The K i values for irreversible inhibition of ( 3[H]QNB binding were 8 × 10 −7, 1 × 10 −7, and 2 × 10-'M for DMBAB, BABC, and IABC, respectively, and for [ 3H]MCC binding, 8 × 10 −5, 1 × 10 −5, and 2 × 10 −5M, respectively. Although DMBAB was found to inhibit the QNB-induced hyperactivity in mice, it did not antagonize the toxic or other pharmacologic effects of oxotremorine. Structure-activity studies with various nonaffinity analogues of the 4-aminobenzoate ester of dimethylaminoethanol and choline revealed that removal of the NH 2; moiety from the phenyl group increased affinity for the muscarinic but not the nicotinic cholinergic site, and quaternization of the ester side chain greatly increased affinity for the muscarinic site. Dimethylation of NH 2 in 4-aminobenzoylcholine decreased the affinity for both cholinergic sites. Replacement of NH 2 by NO 2 increased affinity for the muscarinic but not the nicotinic site, whereas quaternization of the 4-nitrobenzoyl ester markedly increased affinity for the nicotinic site while diminishing affinity for the muscarinic site. The findings indicate that DMBAB and its analogues are useful affinity ligands for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nanomolar concentration range.

Heterogeneous binding of [3H]4‐DAMP to muscarinic cholinergic sites in the rat brain: Evidence from membrane binding and autoradiographic studies

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.

The effects of maternal ethanol exposure on neurotransmission and second messenger systems: a quantitative autoradiographic study in the rat brain

The effects of maternal ethanol exposure on neurotransmission and second messenger systems were examined in rats using histochemistry and in vitro autoradiography. Thirty % ethanol was administered to pregnant rats from gestational day 7 to the day of delivery. Quantitative autoradiography was used to map muscarinic cholinergic, dopamine D 2, adenosine A 1 and inositol 1,4,5-trisphosphate binding sites, as well as to localize adenylate cyclase and protein kinase C. We found no difference in the patterns of staining with acetylcholinesterase and Timm's stain between control and prenatally ethanol-exposed rats on postnatal day (PN) 30. In the ethanol-exposed rats, [ 3H]forskolin binding sites were increased during early development in the CA1 subfield of the hippocampus and the occipital cortex; [ 3H]phorbol ester binding sites were increased in the cortex, striatum, and hippocampus; hippocampal muscarinic cholinergic sites were increased on PN4 and 30; adenosine A 1 binding was reduced on PN10 in most regions examined, but was increased in the CA1 subfield on PN30; dopamine D 2 receptor levels were significantly reduced on PN30 in the striatum; and IP 3 receptors were decreased in most regions studied, but particularly in the cerebellum. Thus, some of these changes were transient and others were long-lasting. Although histophathological abnormalities were minimal, the alterations of binding sites in the cerebellum (the coordination center) and in the hippocampus (related to memory and learning) that were detected may contribute to the behavioral and mental deterioration seen in the fetal alcohol syndrome.

.alpha.-Bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)

Regional development of muscarinic cholinergic binding sites in the prenatal rat brain

The ontogeny of muscarinic cholinergic binding sites was studied in rat fetal central nervous system by in vitro autoradiographic techniques using [ 3H] N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 μM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic cholinergic sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell populations. High receptor densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.

The Binding Characteristics of Cholinergic Sites in Rabbit Spermatozoa

Binding of neurotrophic ligands to rabbit spermatozoa was studied. Nicotinic cholinergic antagonists, [3H]alpha-bungarotoxin and [3H]dihydro-beta-erythroidine (DE), bound with high affinity to different sites in the tails of rabbit spermatozoa with the former binding to 10,207 sites/cell and the latter to 562 sites/cell. alpha-Bungarotoxin and DE sites resemble nicotinic sites in brain in binding affinity and specificity. [3H]Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist, also bound with high affinity to a single class of sites located in the heads and tails of rabbit spermatozoa. The binding characteristics of the sperm muscarinic site are similar to muscarinic sites in both innervated and noninnervated cells. Rabbit spermatozoa incubated for 16-18 h in a medium which supported motility for an extended period possessed fewer binding sites than nonincubated spermatozoa for [3H] alpha-bungarotoxin and [3H]QNB and the KD for the latter ligand was also lower. Ligands specific for the kappa and delta opiate receptors showed no affinity for rabbit spermatozoa.

Selectivity of cimetidine and ranitidine actions at some cholinergic sites in the anaesthetised cat

Selectivity of cimetidine and ranitidine actions at some cholinergic sites in the anaesthetised cat

Lithium but not cholinergic ligands influence guanylate cyclase activity in intact human lymphocytes

The presence of guanylate cyclase in intact circulating human lymphocytes and the sensitivity of this enzyme to stimulation by sodium nitroprusside could be confirmed. However, in contrast to other observations all attempts failed to stimulate the enzyme by cholinergic agonists, despite the use of M1 as well as M2 selective agonists. These findings do not support the assumption that cholinergic recognition sites on human lymphocytes described by many groups are part of a functioning muscarinic transducing mechanism. While several other neuroreceptor agonists were also unable to affect lymphocyte guanylate cyclase activity, lithium was found to potently inhibit the stimulation of guanylate cyclase by sodium nitroprusside at an intracellular concentration close to the therapeutic plasma levels. It is suggested that the effects of lithium on guanylate cyclase activity in human lymphocytes could be related to a possible mechanism of action of lithium in affective disorders.