Effect of substance P on nicotine-induced desensitization of cultured bovine adrenal chromaffin cells: possible receptor subtypes

The neuropeptide substance P (SP) has been reassessed for its ability to modify nicotine-induced catecholamine secretion from cultured bovine, adrenal chromaffin cells. SP exhibited biphasic effects in its actions of inhibiting the nicotinic secretory response and protecting against desensitization. At low concentrations, up to 3 microM, SP partially inhibited or partially protected the nicotine response by 15-20%, and at high concentrations (30 microM), SP markedly inhibited or markedly protected the nicotinic response by 80 or 92%, respectively. The SP antagonist (D-Arg1-D-Pro2-D-Trp7,9-Leu11-SP) completely blocked both effects produced by low concentrations of SP, but not those produced by high concentrations. It is concluded that SP is more potent at protecting against desensitization than at inhibiting the nicotinic response and that SP might modulate CA release through activation of two receptor subtypes.     

Surface and intracellular nicotinic receptors expressed in intact adrenal chromaffin cells: direct measurements using [3H]epibatidine

The presence and importance of assembled, intracellular neuronal nicotinic acetylcholine receptors (nAChRs) has not been established in native systems. In these studies [3H]epibatidine binding techniques were used to characterize surface and intracellular sites expressed in intact bovine adrenal chromaffin cells in culture. Permeant (300 microM nicotine) and impermeant (5 mM carbachol) cholinergic agents were used to define specific [3H]epibatidine binding to total (surface and intracellular) sites and surface sites, respectively. Intracellular [3H]epibatidine binding sites were characterized after eliminating surface binding sites via alkylation. Equilibrium binding to all sites was reached within 30 min at room temperature. Homologous (epibatidine) competition experiments on total (surface and intracellular) binding sites demonstrated a significant fraction of the high affinity sites were localized to intracellular compartments. Saturation binding assays to surface and intracellular sites revealed K(d) values of 1.9+/-1.1 and 3.6+/-1.9 nM, respectively. These binding studies document the existence of a significant population of high affinity, intracellular binding sites in native neuronal cells and support their characterization as assembled, alpha3beta4* nAChRs. Although the intracellular nAChRs represent approximately 70% of the total, high-affinity nAChRs expressed in cultured chromaffin cells, they do not appear to be involved in functional recovery after nAChR down-regulation.     

The interaction of κ-bungarotoxin with the nicotinic receptor of bovine chromaffin cells

Whole-cell recording techniques were used to examine acetylcholine-induced nicotinic currents in isolated bovinei chromaffin cells. The effects on these currents of κ-bungarotoxin, a snake venon κ-neurotoxin, were tested. Exposure of cells to κ-bungarotoxin (600 nM for 40 min) produced a prolonged blockade of nicotinic currents. The mechanism of this blockade was examined in several ways. Firstly, the pre-exposure of cells to trimetaphan, a competitive nicotinic antagonist, protected against the action of subsequent additions of κ-bungarotoxin. Secondly, voltage-clamp measurements indicated that the degree of blockade produced by κ-bungarotoxin was independent of cell membrane potential. Unlike (+)-tubocurarine, κ-bungarotoxin had no direct agonist effects on nicotinic receptors. It is concluded from the present functional studies and from previously reported binding studies that κ-bungarotoxin blocks nicotinic responses in bovine chromaffin cells by binding to regions overlying acetylcholine sites on nicotinic receptors.     

Lead modulation of the neuronal nicotinic acetylcholine receptor in PC12 cells

Lead is known to modulate several ligand- and voltage-gated ion channels, including the nicotinic acetylcholine receptor (AChR) channel. We examined the effects of lead on the nicotinic AChR in rat clonal phaeochromocytoma PC12 cells using whole-cell and single-channel patch-clamp techniques to clarify the detailed mechanism of action. Lead suppressed acetylcholine-induced currents in a dose-dependent manner with an EC₅₀ value of 37 μM and a Hill coefficient of 0.82. At the single-channel level, 1–10 μM lead shortened the opening and burst durations, and increased the duration of mean closed time. The open probability was significantly decreased by lead. These changes of single-channel kinetics result in a significant decrease in the total charge carried through the open AChR channels explaining the suppressive effect of lead on acetylcholine-induced whole-cell currents. © 1997 Elsevier Science B.V. All rights reserved.     

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.     

Alpha7 nicotinic acetylcholine receptors targeted by cholinergic developmental neurotoxicants: nicotine and chlorpyrifos

Alpha7 nicotinic acetylcholine receptors (nAChRs) play a role in axonogenesis, synaptogenesis and synaptic plasticity, and are therefore potential targets for developmental neurotoxicants. We administered nicotine to neonatal rats during discrete periods spanning the onset and peak of axonogenesis/synaptogenesis, focusing on three brain regions with disparate distributions of cell bodies and neural projections: brainstem, forebrain and cerebellum. Nicotine treatment on postnatal days (PN) 1-4 had little or no effect on alpha7 nAChRs but treatment during the second (PN11-14) or third (PN21-24) weeks elicited significant decrements in receptor expression in brainstem and cerebellum, regions containing cell bodies that project to the forebrain. Exposure to chlorpyrifos, a neurotoxicant pesticide that acts partially through cholinergic mechanisms, also elicited deficits in alpha7 nAChRs during the second postnatal week but not the first week. For both nicotine and chlorpyrifos, the effects on alpha7 nAChRs were distinct from those on the alpha4beta2 subtype. Continuous prenatal nicotine exposure, which elicits subsequent, postnatal deficits in axonogenesis and synaptogenesis, also produced delayed-onset changes in alpha7 nAChRs, characterized by reductions in the forebrain and upregulation in the brainstem and cerebellum, a pattern consistent with impaired axonogenesis/synaptogenesis and reactive sprouting. Males were more sensitive to the persistent effects of prenatal nicotine exposure on alpha7 nAChRs, a pattern that mimics neurobehavioral deficits resulting from this treatment. The present findings reinforce the mechanistic involvement of alpha7 nAChRs in the actions of developmental neurotoxicants, and its biomarker potential for neuroteratogens that target neuritic outgrowth.     

Characterization of nicotinic receptors in immortalized hippocampal neurons

Nicotinic acetylcholine receptors, particularly nicotinic a-bungarotoxin (a-BGT) receptors, are present in relatively high concentrations in rat hippocampus. Because of the difficulties encountered in studying receptors using primary cells in culture, especially for biochemical work, we investigated the possibility of using an immortalized cell line from embryonic rat hippocampus (H19-7). RNase protection assays show that α4, α7 and β2 neuronal nicotinic receptor subunit mRNAs are present in differentiated but not undifferentiated H19-7 cells, while α2, α3, α5 and β3 subunit mRNAs were not detectable under either condition. In line with these results, the present data demonstrate that the H19-7 cells express cell surface nicotinic a-BGT binding sites, which were maximal after seven days of differentiation in culture. The receptors were saturable, of high affinity (K_d = 1.30 nM and B_max = 11.70 fmol/10⁵ cells) and had a pharmacological profile similar to that observed for CNS a-BGT receptors. On the other hand, although α4 and β2 neuronal nicotinic subunit mRNAs were present in differentiated H19-7 cells, no [3H]cytisine binding was observed. Because immortalized cell lines have the advantage that they provide a limitless supply of cells as compared to primary cell cultures, but yet are not malignant in origin, the present results may suggest that the H19-7 immortalized hippocampul cell line represent a useful CNS model system for examining α-BGT nicotinic receptors.     

Human cortical neurons contain both nicotinic and muscarinic acetylcholine receptors: an immunocytochemical double-labeling study

Using immunofluorescence histochemistry, in the human cerebral cortex neurons immunoreactive for both nicotinic and muscarinic acetylcholine receptor proteins could be demonstrated. Vibratome sections of biopsy and autopsy specimens of human temporal and occipital lobes were incubated with monoclonal antibodies specific for muscarinic (M 35) and nicotinic (WF 6) acetylcholine receptor protein. Immunoreactive sites were visualized using a biotin-streptavidin-phycoerythrin system (M 35, red fluorescence) and fluorescein-conjugated immunoglobulins (WF 6, green fluorescence). Immunofluorescence of both antibodies was preponderant in pyramidal neurons located in layers II/III and V and their apical dendrites. Some round and ovoid immunolabeled cells were encountered in layers VI and IV. About 30% of the cholinoceptive cortical neurons, in particular the pyramidal cells, displayed immunoreactivity for both receptor types. The present investigation shows a subpopulation of human cortical neurons to contain both nicotinic and muscarinic receptors. The coexistence of acetylcholine receptors may provide the morphological basis of simultaneous impact of acetylcholine on both receptor types in the same neuron of the human cerebral cortex.     

A postmortem study of brain nicotinic receptors in Parkinson's and Alzheimer's disease

Brain nicotinic receptors were studied in the frontal cortex, temporal cortex, hippocampus and caudate nucleus in patients with Parkinson's disease (PD), Alzheimer's disease (AD) and controls. The B_max and K_d values of (−)-[³H]nicotine binding were determined with a Scatchard analysis. The number of nicotinic receptors declined both in PD and in AD patients in all brain areas examined. The K_d values were unchanged. There was a negative correlation between the degree of dementia in PD patients and the number of nicotinic receptors in the frontal cortex. A similar correlation was seen between the muscarinic/nicotinic receptor ratio in the frontal cortex and the degree of dementia in PD patients. The present findings indicate that nicotinic receptors are affected not only in AD, but also in PD and that dysfunction of the cholinergic system in the frontal cortex is involved in the dementia process in PD.     

ICV STZ induced impairment in memory and neuronal mitochondrial function: A protective role of nicotinic receptor

The present study was planned to evaluate the cholinergic influence on mitochondrial activity and neurodegeneration associated with impaired memory in intracerebroventricular (ICV) streptozotocin (STZ) treated rats. STZ (3 mg/kg), administered ICV twice with an interval of 48 h between the two doses, showed significant impairment in spatial memory tested by water maze test 14 days after first dose without altering blood glucose level and locomotor activity. Animals were sacrificed on 21st day of ICV administration. STZ significantly increased malondialdehyde (MDA), reactive oxygen species (ROS), Ca²⁺ ion influx, caspase-3 activity and decreased glutathione (GSH) level. Acetylcholinesterase inhibitors tacrine and donepezil (5 mg/kg, PO) pretreatment significantly prevented STZ induced memory deficit, oxidative stress, Ca²⁺ influx and caspase-3 activity. Carbachol, a muscarinic cholinergic agonist (0.01 mg/kg, SC) did not show any significant effect on ROS generation, Ca²⁺ ion influx and caspase-3 activity. While nicotinic cholinergic agonist, nicotine, significantly attenuated ICV STZ induced mitochondrial dysfunction and caspase-3 activity. The results indicate that instead of muscarinic receptors nicotinic receptors may be involved in neuroprotection by maintaining mitochondrial functions.     

Neuroprotective action of donepezil mediated by neuronal nicotinic receptors

AChE inhibitors used in the treatment in Alzheimer’s disease such as donepezil are effective in preventing glutamate neurotoxicity. In primary cultures of the cortical neurons, donepezil prevents glutamate neurotoxicity when the drug is applied 8–24 hr prior to glutamate exposure. Neuroprotective effect of donepezil is antagonized by mecamylamine, dihydro‐β‐erythoridine and metyllcaconitine. Prolonged (more than 4 days) exposure of the cultures to donepezil induces an increase in the nicotine‐induced Ca²⁺ influx and number of neurons expressing α4 and α7 subunits of nicotinic receptors. Donepezil also prevents apoptotic neuronal death induced by glutamate. Inhibitors for non‐receptor type tyrosine kinase, Fyn and janus‐activated kinase 2, suppress the neuroprotective effect of donepezil. Neuroprotective effect of donepezil is also suppressed by a phosphatidylinositol 3‐kinase (PI3K) inhibitor. Phosphorylation level of Akt, an effector of PI3K, and the expression level of Bcl‐2 increase with donepezil. These results suggest that donepezil prevents glutamate neurotoxicity through α4‐ and α7‐nicotinic acetylcholine receptors (nAChRs), followed by activation of PI3‐Akt pathway but independent from activation of ion channels associated with nAChRs. α4‐ and α7‐nAChRs may play an important role in promoting survival of cortical neurons under oxidative stress caused by excitotoxicity.     

Possible role of surface potential in the gating mechanism of Ca channels in cat adrenal chromaffin cells: studies with fura-2 microfluorometry

The cytosolic free Ca²⁺ concentration ([Ca]_in) in isolated cat chromaffin cells was measured by fura-2 microfluorometry. During 30 mM KCl depolarization or sucrose substitution for NaCl, a reduction in external Ca²⁺ concentration under optimal conditions paradoxically caused a rise in [Ca]_in and, in separate experiments, in catecholamine secretion. The results support a previously suggested role of surface potentials in the gating mechanism of Ca²⁺ channels.     

Modulation of the nicotinic α-bungarotoxin site in chromaffin cells in culture by a factor(s) endogenous to neuronal tissue

An endogenous factor(s) which affects the in vitro binding of α-bungarotoxin (α-BGT) to rat brain membranes has previously been found in brain supernatant. This fraction, as well as a partially purified preparation of this material from bovine brain, is here shown to affect the binding of α-BGT to chromaffin cell membranes. To study possible long term effects, the supernatant extract was added to adrenal medullary chromaffin cells in culture. The cells were incubated for several days and at the end of this time, the medium bathing the cells, which contained the endogenous factor(s), was removed and α-BGT binding to the cells measured. Binding to control cultures had shown that α-BGT bound to the chromaffin cells in a saturable manner, with high affinity (K_d = 1.5 nM) and the specificity of a nicotinic receptor ligand. After incubation of the cells with supernatant factor, a marked decline in the number of a α-BGT binding sites was observed with no change in affinity. This does not appear to be due to a detrimental effect on the cells as cell number did not appear to be decreased in the cultures preincubated with the supernatant extract and the DNA and protein content were similar in the control and treated cultures. The possibility that there was some non-specific detrimental effect to the chromaffin cell membrane was considered; however, the stimulated release of noradrenaline from the cells was not affected by treatment of the cultures in the presence of the supernatant fractions. In addition, tyrosine hydroxylase activity was significantly increased in the treated cultures. d-Tubocurarine, an antagonist at the acetylcholine receptor, caused an increase in α-BGT binding after 7 days of treatment, while the agonist nicotine and choline had no effect. These results suggest that in brain supernatant there may exist an endogenous factor(s), which may function in the regulation of the nicotinic-like α-BGT receptors in neuronal cell.     

Colocalization of muscarinic and nicotinic receptors in cholinoceptive neurons of the suprachiasmatic region in young and aged rats

In the present study muscarinic and nicotinic cholinergic receptors in the SCN region were demonstrated and analyzed, employing monoclonal antibodies to purified muscarinic and nicotinic cholinergic receptor proteins. A near-total colocalization of the two acetylcholine receptor subclasses in cholinoceptive neurons of the SCN area was found. The antibodies applied to aging rat brain (at 30–34 months) revealed a clear decrease in immunoreactivity in senescence albeit with a high level of individual variability. Furthermore, in 8 out of 10 aged animals examined a considerable increase of astrocytes possessing muscarinic cholinergic receptors was observed.     

Characterization of substance P and somatostatin receptors on adrenal chromaffin cells using structural analogues

Substance P (SP) and somatostatin (SRIF) are known to inhibit the nicotine-induced release of catecholamines (CAs) from isolated adrenal chromaffin cells in culture^22,24. In order to characterize the receptors mediating this action, we have tested several SP and SRIF analogues for their effects on release of [³H]l-norepinephrine ([³H]NE) from chromaffin cell cultures. SP-free acid and a series of 11 SP analogues, in which each amino acid of SP is replaced in turn byl-alanine, all inhibited the nicotine-induced release of [³H]NE from these cultures. The rank order of potency of the analogues for this action was similar to their reported order of potency in other SP-responsive tissues. The least potent were SP-free acid, [Ala⁷]-SP, [Ala¹⁰]-SP, [Ala⁸]-SP and [Ala¹¹]-SP, while the potencies of the Ala 1 to 6 analogues and [Ala⁹]-SP were closer to that of SP. [Leu⁷]- and [Leu^7,8]-SP had potencies similar to that of SP. SP itself had no effect on basal [³H]NE release. The results suggest that adrenal chromaffin cells possess a specific SP receptor mediating inhibition of agonist-induced CA release and that the binding site of this receptor shares similar structural requirements with the binding site of the SP receptor on other tissues.Several SRIF analogues, which have been previously shown to be more potent than native SRIF at selective SRIF receptors^{2,3,31, 35}, were compared to SRIF for effects on [³H]NE release from chromaffin cell cultures. These analogues were found to be active but less potent than SRIF in inhibiting nicotine-induced [³H]NE release from these cultures, suggesting that the site mediating this action differs in its structural requirements from the SRIF receptor found in some other tissues.     

Cholinergic nicotinic and muscarinic receptors in dementia of Alzheimer, Parkinson and Lewy body types

Summary Cholinergic nicotinic and muscarinic receptor binding were measured in post mortem human brain tissue, using low (nM) concentrations of (³H)-nicotine to detect predominately the high affinity nicotinic site and (³H)-N-methylscopolamine in the presence and absence of 3×10^–4 M carbachol to measure both the low and high affinity agonist subtypes of the muscarinic receptor group. Consistent with most previous reports, the nicotinic but not muscarinic binding was reduced in the different forms of dementia associated with cortical cholinergic deficits, including Alzheimer's and Parkinson's disease, senile dementia of Lewy body type (SDLT) and Down's syndrome (over 50 years). Analysis of (³H)-nicotine binding displaced by a range of carbachol concentrations (10^–9–10^–3 M) indicated 2 binding sites for nicotine and that the high affinity rather than low affinity site was reduced in Alzheimer's disease. In all 3 cortical areas investigated (temporal, parietal and occipital) there were increases in the low affinity muscarinic site in Parkinson's disease and SDLT but not Alzheimer's disease or middle-aged Down's syndrome. This observation raised the question of whether the presence of neurofibrilalry tangles (evident in the latter but not former 2 disorders) is incompatible with denervation-induced muscarinic supersensitivity in cholinoceptive neurons which include cortical pyramids generally affeced by tangle formation.     

[I]Epibatidine-labelled nicotinic receptors in the extended striatum and cerebral cortex: lack of association with serotonergic afferents

In rat extended striatum, most nicotinic cholinoceptors are likely to be presynaptic. A previous report suggested that DA and 5-HT afferents each account for at least 30% of nicotinic binding sites in the striatum. To explore this question further, rats received unilateral infusions of the neurotoxins 5,7-dihydroxytryptamine, 6-hydroxydopamine or vehicle into the medial forebrain bundle, and were sacrificed 3 weeks later. Denervation was quantified by [¹²⁵I]RTI-55 autoradiography, using separate assay conditions that revealed DA and 5-HT transporters (i.e. DAT and SERT). Nicotinic cholinoceptors were quantified by [¹²⁵I]epibatidine autoradiography. Infusion of 6-hydroxydopamine depleted DAT but not SERT labelling in all striatal areas (i.e. caudate-putamen, nucleus accumbens core and shell, olfactory tubercle). The serotonergic neurotoxin 5,7-dihydroxytryptamine depleted SERT and, to a lesser extent, DAT labelling. Both neurotoxins reduced [¹²⁵I]epibatidine binding in striatal areas. Multiple linear regression analysis showed that these reductions in [¹²⁵I]epibatidine binding were entirely associated with loss of DAT rather than SERT. The DAT-associated proportion of total [¹²⁵I]epibatidine binding was 36±2% (caudate-putamen), 28±3% (accumbens core), 27±4% (accumbens shell) and 44±5% (olfactory tubercle). Cortical [¹²⁵I]epibatidine binding was unaltered by 5,7-dihydroxytryptamine lesions that reduced SERT labelling by 46 to 73%. In all brain areas, even small (3.4 to 8.8%) SERT-associated reductions in [¹²⁵I]epibatidine binding would have been detected as statistically significant. In conclusion, we report the failure to detect nAChRs on 5-HT terminals in extended striatum or cerebral cortex, using a sensitive [¹²⁵I]epibatidine autoradiographic assay.     

Cell therapy of pain: Characterization of human fetal chromaffin cells at early adrenal medulla development

Adult adrenal chromaffin cells are being utilized for therapeutic transplantation. With the prospect of using fetal chromaffin cells in pain therapy, we studied their phenotype, proliferative power, function, and growth in vitro and in situ in order to determine the optimal time for implantation. Between 7 and 10 gestational weeks (GW), we isolated, in vitro, two types of chromaffin cells with a noradrenergic phenotype akin to that observed, in situ. Among the adherent chromaffin cells first observed in vitro, only a few samples expressed met-enkephalin, whereas almost all the neurosphere-like colonies, which appeared later, expressed it. However, neither of the two types of populations expressed an adrenergic phenotype in line with that observed in situ. At the upper limits of the voluntary abortion period authorized in France, this phenotype (12 GW) and met-enkephalin expression (13 GW) were evidenced in situ. For the first time in man, we demonstrate the secretion of noradrenaline in vitro by the two populations of cells. Consistent with this result, we also noted dopamine beta hydroxylase (DbetaH) mRNA expression in vitro and in situ within this period. These observations on the expression of these biological factors indicate that 9-10 GW would be the best stage for sampling these cells for preclinical transplantation experiments.